(-)-Epigallocatechin Gallate Derivatives For Inhibiting Proteasome

ABSTRACT

(−)-EGCG, the most abundant catechin, was found to be chemopreventive and anticancer agent. However, (−)-EGCG has at least one limitation: it gives poor bioavailability. This invention provides compounds of generally formulae below, wherein R 11 , R 12 , R 13 , R 21 , R 22 , R 2 , R 3 , and R 4  are each independently selected from the group consisting of —H, and C 1  to C 10  acyloxyl group; and R 5  is selected from the group consisting of —H, C 1 -C 10 -alkyl, C 2 -C 10 -alkenyl, C 2 -C 10 -alkynyl, C 3 -C 7 -cycloalkyl, phenyl, benzyl and C 3  -C 7  -cycloalkenyl, whereas each of the last mentioned 7 groups can be substituted with any combination of one to six halogen atoms; at least one of R 11 , R 12 , R 13 , R 21 , R 22 , R 2 , R 3  and R 4  is —H, which were found to be more potent than their non-protected counterparts, which can be used as proteasome inhibitors to reduce tumor cell growth.

FIELD OF THE INVENTION

This invention relates to derivates of (−)-epigallocatechin gallate,particularly for use as proteasome inhibitors and/or for inhibition ofcancer cell growth.

BACKGROUND OF THE INVENTION

The polyphenols found in green tea extracts are (−)-epicatechin (EC),(−)-epigallocatechin (EGC), (−)-epicatechin-3-gallate (ECG) and(−)-epigallocatechin-3-gallate (EGCG). In particular, (−)-EGCG, the mostabundant catechin, was found to be chemopreventive and anticancer agentamong the green tea catechins (GTCs) (4. Fujiki, H. J Cancer Res ClinOncol. 999, 125, 589-97).

Proteasome is a large protein complex with multicatalytic activitiesthat are responsible for the degradation of not only obsolete andmisfolded proteins, but also regulatory proteins involved in cell cycleand apoptosis. In proteasome-dependent proteolysis, ubiquitin is firstconjugated to the substrate, followed by degradation of the substrateand recycling of the amino acids and ubiquitin. Theubiquitin/proteasome-dependent degradation pathway plays an essentialrole in up-regulation of cell proliferation, down-regulation of celldeath, and development of drug resistance in human tumor cells.Therefore, proteasome inhibitors show great potential as novelanticancer drugs (Dou, Q. P.; Li, B. Drug Resist Update 1999, 2,215-23). It has been shown that natural (−)-EGCG and syntheticallyderived (+)-EGCG are potent inhibitors of the proteasomal chymotrypsinactivity, leading to growth arrest and/or apoptosis (Smith, D. M.; Wang,Z.; Kazi, A.; Li, L.; Chan, T. H.; Dou, Q. P. Mol Med 2002, 8: 382-92.).US patent publication no. 20040110790 (Zaveri et al.) describessynthetic analogs of green tea polyphenols as chemotherapeutic andchemopreventive agents, but the synthesis provided only racemiccompounds, and do not use natural occurring catechins derived from greentea.

The P13K/Akt signaling is a widely known tumor cell survival pathway(Vanhaesebroeck, B.; Alessi, D. R. Biochem J 2000, 346, 561-76).Blocking this pathway is considered as an important mechanism forinhibiting tumor growth. Phosphorylated Akt (p-Akt) is the activatedform of Akt. Once Akt is activated, it can mediate cell cycleprogression by phosphorylation and consequent inhibition of thecyclindependent kinase inhibitor p 27.24 Recently, (−)-EGCG has beenfound to inhibit the Akt kinase activity via reducing thephosphatidylinositol 3-kinase signals in MMTV-Her-2/neu mouse mammarytumor NF639 cells, leading to reduced tumor cell growth (Pianetti, S.;Guo, S.; Kavanagh, K. T.; Sonenshein, G. E. Cancer Res 2002, 62, 652-5).

However, (−)-EGCG has at least one limitation: it gives poorbioavailability. A study by Nakagawa et al. showed that only 0.012% of(−)-EGCG could be absorbed in rats given 56 mg of (−)-EGCG orally(Nakagawa, K.; Miyazawa, T. Anal Biochem. 1997, 248, 41-9). This lowabsorption was thought to be due to the poor stability of (−)-EGCG inneutral or alkaline solutions. As pH value of the intestine and bodyfluid is neutral or slightly alkaline, GTCs will be unstable inside thehuman body, thus leading to reduced bioavailability.

OBJECTS OF THE INVENTION

Therefore, it is an object of this invention to provide a (−)-EGCGderivative that is able to resolve at least one or more of the problemsas set forth in the prior art. As a minimum, it is an object of thisinvention to provide the public with a useful choice.

SUMMARY OF THE INVENTION

Accordingly, this invention provides a compound for inhibitingproteasome having the formula:

-   -   wherein    -   R₁₁, R₁₂, R₁₃, R₂₁, R₂₂, R₂, R₃, and R₄ are each independently        selected from the group consisting of —H, and C₁ to C₁₀ acyloxyl        group; and    -   R₅ is selected from the group consisting of —H, C₁-C₁₀-alkyl,        C₂-C₁₀-alkenyl, C₂-C₁₀-alkynyl, C₃-C₇-cycloalkyl, phenyl, benzyl        and C₃-C₇-cycloalkenyl, whereas each of the last mentioned 7        groups can be substituted with any combination of one to six        halogen atoms;    -   at least one of R₁₁, R₁₂, R₁₃, R₂₁, R₂₂, R₂, R₃, and R₄ is C₁ to        C₁₀ acyloxyl group; and    -   at least one of R₁₁, R₁₂, R₁₃, R₂₁, R₂₂, R₂, R₃, and R₄ is —H.

Preferably each of R₁₁, R₂, and R₄ is —H, and each of R₁₂, R₁₃, R₂₁,R₂₂, and R₃ is an acetate or benzoate group.

Optionally, R₁₁, is —H, and each of R₁₂, R₁₃, R₂₁, R₂₂, R₂, R₃, and R₄is an acetate or benzoate group.

Additionally each of R₁₁, R₁₃, R₂, and R₄ is —H, and each of R₁₂, R₂₁,R₂₂, and R₃ is an acetate or benzoate group.

Optionally, each of R₁₁, and R₁₃ is —H, and each of R₁₂, R₂₁, R₂₂, R₂,R₃, and R₄ is an acetate or benzoate group.

Each of R₁₁, R₁₂, and R₁₃ can be —H, and each of R₂₁, R₂₂, R₂, R₃, andR₄ can be an acetate or benzoate group.

In one embodiment of the above compound this invention, R₅ is —H, andeach of R₁₁, R₁₂, R₁₃, R₂₁, and R₂₂ is an acetate group. This particularembodiment also provides the following three variations:

-   -   R₂ is an acetate group, and each of R₃ and R₄ is —H;    -   R₃ is an acetate group, and each of R₂ and R₄ is —H; or    -   each of R₂ and R₄ is an acetate group, and R₃ is —H.

It is another aspect of this invention to provide a method of reducingtumor cell growth including the step of administering an effectiveamount of a compound having the formula:

-   -   wherein    -   R₁₁, R₁₂, R₁₃, R₂₁, R₂₂, R₂, R₃, and R₄ are each independently        selected from the group consisting of —H, and C₁ to C₁₀ acyloxyl        group; and    -   R₅ is selected from the group consisting of —H, C₁-C₁₀-alkyl,        C₂-C₁₀-alkenyl, C₂-C₁₀-alkynyl, C₃-C₇-cycloalkyl, phenyl, benzyl        and C₃-C₇-cycloalkenyl, whereas each of the last mentioned 7        groups can be substituted with any combination of one to six        halogen atoms; and    -   at least one of R₁₁, R₁₂, R₁₃, R₂₁, R₂₂, R₂, R₃, and R₄ is C₁ to        C₁₀ acyloxyl group.

It is yet another aspect of this invention to provide a use of acompound of having the formula:

-   -   wherein    -   R₁₁, R₁₂, R₁₃, R₂₁, R₂₂, R₂, R₃, and R₄ are each independently        selected from the group consisting of —H, and C₁ to C₁₀ acyloxyl        group; and    -   R₅ is selected from the group consisting of —H, C₁-C₁₀-alkyl,        C₂-C₁₀-alkenyl, C₂-C₁₀-alkynyl, C₃-C₇-cycloalkyl, phenyl, benzyl        and C₃-C₇-cycloalkenyl, whereas each of the last mentioned 7        groups can be substituted with any combination of one to six        halogen atoms; and    -   least one of R₁₁, R₁₂, R₁₃, R₂₁, R₂₂, R₂, R₃, and R₄ is C₁ to        C₁₀ acyloxyl group in the manufacturing of a medicament for        reducing tumor cell growth.

This invention also provides a compound for inhibiting proteasome havingthe formula:

-   -   wherein    -   R₁ is —H;    -   R₂, R₃, and R₄ are each independently selected from the group        consisting of —H and —OH; and    -   at least one of R₂, R₃, and R₄ is —H.

Preferably, R₂ can be —OH, and R₃═R₄═—H. Optionally, R₃ may be —OH, andR₂═R₄═—H; or R₃ may be —H, and R₂═R₄═—OH.

This invention also provides a method of reducing tumor cell growthincluding the step of administering an effective amount of a compoundhaving the formula:

-   -   wherein    -   R₁ is —H;    -   R₂, R₃, and R₄ are each independently selected from the group        consisting of —H and —OH; and    -   if R₂═R₃═R₄, then R₂ is not —OH.

It is another aspect of this invention to provide the use of a compoundof having the formula:

-   -   wherein    -   R₁ is —H;    -   R₂, R₃, and R₄ are each independently selected from the group        consisting of —H and —OH; and    -   if R₂═R₃═R₄, then R₂ is not —OH    -   in the manufacturing of a medicament for reducing tumor cell        growth.

BRIEF DESCRIPTION OF THE DRAWINGS

Preferred embodiments of the present invention will now be explained byway of example and with reference to the accompanying drawings in which:

FIG. 1 shows the structures of the (−)-EGCG, and examples of the(−)-EGCG derivatives of this invention;

FIG. 2 shows the degradation curve of (−)-EGCG and 1;

FIG. 3 shows the time-course results of peracetate EGCG (1) in culturemedium with the presence of vitamin C (area vs time). Compound 1: ♦;compound A (di-acetate): x; compound B (mono-acetate): ▴; EGCG: ▪;

FIG. 4 shows the time-course results of peracetate EGCG (1) in culturemedium with the presence of vitamin C with the addition of lysate (areavs time). Compound 1: ♦; compound A (di-acetate): x; compound B(mono-acetate): ▴; EGCG: ▪;

FIG. 5 shows the inhibition of the chymotrypsin-like activity of thepurified 20S proteasome by 1 and (−)-EGCG;

FIG. 6 shows (a) the inhibition of Proteasome Actvitiy by 1 and (−)-EGCGin vivo; (b) Western blot assay of ubiquitin after treatment with 1 and(−)-EGCG;

FIG. 7 shows the amount of p-Akt levels with 1 and (−)-EGCG treatment;

FIG. 8 shows the cell viability in Jurkat cells treated with 1 and(−)-EGCG;

FIG. 9 shows the results of treating Jurkat cells with 25 μM of eachindicated polyphenol for 4 h (A), up to 8 (C), or 24 h (B), or of LNCaPcells treated with 25 μM of indicated compound for 24 h (D), followed byWestern blot analysis using specific antibodies to Ubiquitin, Bax, IκBα,p27 and Actin. The bands indicated by an arrow are possibleubiquitinated forms of Bax and IκBα. A, Lane 4, Ub-IκBα band may beresult of spillage from Lane 5. Data shown are representative from threeindependent experiments;

FIG. 10 shows the results of treating Jurkat T cells (A and B) or VA-13(C and D) cells with 25 μM of indicated polyphenols for 24 h. A, Trypanblue incorporation assay. The data represented are as the mean number ofdead cells over total cell population ±SD. B, Western blot for PARPcleavage. C, Fluorescent microscopy studies of late-stage apoptosisusing a specific antibody to the p85 cleaved PARP fragment conjugated toFITC. Counterstaining with DAPI is used as a control for non-apoptoticcells. Images were obtained with AxioVision software utilizing aninverted fluorescent microscope (Zeiss, Germany). D, Quantification ofapoptotic cells in C by counting the number of apoptotic cells over thetotal number of cells in the same field. Data are mean of duplicateexperiments ±SD;

FIG. 11 shows the effects of synthetic acetylated polyphenols on breastand prostate cancer cells. A, MTT assay. Breast cancer MCF-7 cells weretreated with each indicated compound at 5 or 25 μM for 24 h. B,Morphological changes. Prostate cancer LNCaP cells were treated with 25μM of (−)-EGCG or a protected analog for 24 h, followed by morphologicalassessment. Images were obtained using a phase-contrast microscope at40× magnification (Leica, Germany). C, Soft agar assay. LNCaP cells wereplated in soft agar with the solvent DMSO or 25 pM of (−)-EGCG orprotected analogs. Cells were cultured for 21 days without furtheraddition of drug. Data shown are representative scanned wells fromtriplicate experiments. D, Colonies in C were quantified with anautomated counter and presented as mean values ±SD;

FIG. 12 shows the results of treating normal WI-38 and SV-40-transformedVA-13 cells with 25 μM of indicated compounds for 24 h (A and B) or 36 h(C), or leukemic Jurkat T and non-transformed YT cells were treated witheach compound at 25 μM for 24 h (D). A, Chymotrypsin-like activity ofthe proteasome in intact cells. B and C, Nuclear staining for apoptoticmorphology of both detached and attached cells at 10× (B) or 40× (C)magnification. Missing panels indicate that no detachment of cellsoccurred. D, Western blot analysis using specific antibody to PARP;

FIG. 13 shows the structures of the (−)-EGCG, and examples of the(−)-EGCG peracyloxyl derivatives of this invention. FIGS. 13 a and 13 bshow the intermediates when synthesizing the (−)-EGCG peracyloxylderivatives of this invention;

FIG. 14 shows the chymotrypsin-like activity when Jurkat T cells werepreincubated with the solvent (DMSO), 25 μM of the compounds in FIG. 13;

FIG. 15 shows the accumulation of proteasome target and ubiquitinatedproteins when Jurkat T cells were treated with the solvent (DMSO), and25 μM of the compounds in FIG. 13;

FIG. 16 shows the percentage cell death when Jurkat T cells were treatedwith the solvent (DMSO), and 25 μM of the compounds in FIG. 13;

FIG. 17 shows the activation of caspase-3 when Jurkat T cells weretreated with the solvent (DMSO), and 25 μM of the compounds in FIG. 13;and

FIG. 18 shows the induction of apoptosis in tumor cells when Jurkat Tcells and non-transformed human natural killer (YT) cells were treatedfor 4 h with 10 and 25 μM of the 19*, followed by Western blot analysisusing specific antibodies to IκB-α, PARP and Actin.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

This invention is now described by way of example with reference to thefigures in the following paragraphs.

Objects, features, and aspects of the present invention are disclosed inor are obvious from the following description. It is to be understood byone of ordinary skill in the art that the present discussion is adescription of exemplary embodiments only, and is not intended aslimiting the broader aspects of the present invention, which broaderaspects are embodied in the exemplary constructions.

In this invention, prodrugs form of (−)-EGCG is synthesized thatimproves its bioavailability. The prodrugs exhibit: [i] improvedstability in physiological conditions at a neutral pH; [ii] remainbiologically inactive until enzymatic hydrolysis in vivo, leading to therelease of the parent drug; [iii] and lastly, the promoiety groupspossess low systemic toxicity

Further, three derivatives of (−)-EGCG and their prodrug forms aresynthesized, and surprisingly, are found to have higher potency than thenatural form of (−)-EGCG itself.

The general formula of the derivatives of (−)-EGCG of this invention inalcohol form has the formula:

-   -   wherein    -   R is —H; and    -   R₂, R₃, and R₄ are each independently selected from the group        consisting of —H and —OH.

Of course, when R₂═R₃═R₄, R₂ is —OH, the compound becomes (−)-EGCG, andtherefore is not the subject of this invention.

The general formula of the derivatives of (−)-EGCG of this invention inester form has the formula:

-   -   wherein    -   R₁₁, R₁₂, R₁₃, R₂₁, R₂₂, R₂, R₃, and R₄ are each independently        selected from the group consisting of —H, and C₁ to C₁₀ acyloxyl        group; and    -   R₅ is selected from the group consisting of —H, C₁-C₁₀-alkyl,        C₂-C₁₀-alkenyl, C₂-C₁₀-alkynyl, C₃-C₇-cycloalkyl, phenyl, benzyl        and C₃-C₇-cycloalkenyl, whereas each of the last mentioned 7        groups can be substituted with any combination of one to six        halogen atoms;    -   at least one of R₁₁, R₁₂, R₁₃, R₂₁, R₂₂, R₂, R₃, and R_(4 is C)        ₁ to C₁₀ acyloxyl group; and    -   at least one of R₁₁, R₁₂, R₁₃, R₂₁, R₂₂, R₂, R₃, and R₄ is —H.

In the definitions of the compounds above, collective terms were usedwhich generally represent the following groups:

C₁-C₆ acyl: having the structure —(CO)—R, wherein R is hydrogen orstraight-chain or branched alkyl groups having 1 to 5 carbon atoms, suchas methyl, ethyl, propyl, 1-methylethyl, butyl, 1-methylpropyl,2-methylpropyl, 1,1-dimethylethyl, pentyl, 2-methylbutyl. The alkylgroup R can be partially or fully halogenated”. The term “partially orfully halogenated” is meant to express that in the groups characterizedin this manner the hydrogen atoms may be partially or fully replaced byidentical or different halogen atoms, for example chloromethyl,dichloromethyl, trichloromethyl, fluoromethyl, difluoromethyl,trifluoromethyl, chlorofluoromethyl, dichlorofluoromethyl,chlorodifluoromethyl, 1-fluoroethyl, 2-fluoroethyl, 2,2-difluoroethyl,2,2,2-trifluoroethyl, 2-chloro-2-fluoroethyl,2-chloro-2,2-difluoroethyl, 2,2-dichloro-2-fluoroethyl,2,2,2-trichloroethyl and pentafluoroethyl;

C₁-C₁₀ acyloxyl: having the structure —O—(CO)—R, wherein R can be anyone of —H, C₁-C₉-alkyl, C₂-C₉-alkenyl, C₂-C₉-alkynyl, C₃-C₇-cycloalkyl,phenyl, benzyl and C₃-C₇-cycloalkenyl, whereas each of the lastmentioned 7 groups can be substituted with any straight chain orbranched alkyl groups, such as methyl, ethyl, propyl, 1-methylethyl,butyl, 1-methylpropyl, 2-methylpropyl, 1,1-dimethylethyl, pentyl,2-methylbutyl. The alkyl group R can be partially or fully halogenated”.The term “partially or fully halogenated” is meant to express that inthe groups characterized in this manner the hydrogen atoms may bepartially or fully replaced by identical or different halogen atoms, forexample chloromethyl, dichloromethyl, trichloromethyl, fluoromethyl,difluoromethyl, trifluoromethyl, chlorofluoromethyl,dichlorofluoromethyl, chlorodifluoromethyl, 1-fluoroethyl,2-fluoroethyl, 2,2-difluoroethyl, 2,2,2-trifluoroethyl,2-chloro-2-fluoroethyl, 2-chloro-2,2-difluoroethyl,2,2-dichloro-2-fluoroethyl, 2,2,2-trichloroethyl and pentafluoroethyl.The alkyl group R can be partially or fully substituted by hydroxy, oralkoxy or amino groups, for example, hydroxymethyl, 2-aminoethyl, or3-methoxypropyl groups.

According to this invention, peracetate (−)-EGCG, 1 was synthesized(FIG. 1). 1 was found to be more stable than (−)-EGCG. The prodrug wasbiologically inactive against a purified 20S proteasome, but potentlyinhibited the proteasome in intact tumor cells. Furthermore,administration of the prodrug, but not its parent compound, to intacttumor cells resulted in the loss of phosphorlyated Akt (p-Akt),indicating inactivation of this cancer-associated kinase. Finally,treatment of leukemia Jurkat T cells with 1 induced cell death.

In order to evaluate whether other peractete protected tea polyphenolspossessed greater bioactivity than their unprotected parent, severalsynthetic analogs to (−)-EGCG that possess deletions of the hydroxylgroups on the gallate ring were synthesized. Additionally, to enhancethe stability of the molecules, the hydroxyl groups were converted toacetate or benzoate groups to create a prodrug. Surprisingly, theprotected analogs were found to be more potent proteasome inhibitors inintact tumor cells than their unprotected counterparts.

The synthesis and characterization of the compounds of this inventionwill be detailed in the following sections.

Materials and Methods Reagents

Fetal Bovine Serum was purchased from Tissue Culture Biologicals(Tulare, Calif.). Mixture of penicillin-streptomycin-1-Glutaxnine, RPMI,and DMEM are from Invitrogen (Carlsbad, Calif.). Dimethyl sulfoxide(DMSO), N-acetyl-L-cysteine (NAC), Hoechst 33342,3-((4,5)-dimethylthiazol-2-yl)-2,5-diphenylteolium bromide (MTT), bovineserum albumin (BSA), and (−)-EGCG were purchased from Sigma (St. Louis,Mo.). Suc-Leu-Leu-Val-Tyr-AMC (for the proteasomal chyrnotrypsin-likeactivity) was obtained from Biomol (Plymouth Meeting, Pa.). Purified 20sproteasome from rabbit was acquired from Boston Biochem (Cambridge,Mass.). Arnplex Red H202 assay kit was purchased fiom Molecular Probes(Eugene, Oreg.). Monoclonal antibodies to Bax (H280) and Ubiquitin(P4D1), and polyclonal antibodies to IKB-a (C15), and Actin (C11) aswell as anti-goat, anti-rabbit, and anti-mouse IgG-horseradishperoxidase were purchased from Santa Cruz Biotechnology (Santa Cruz,Calif.). Monoclonal antibody to p27 (554069) was purchased from BDBiosciences (San Diego, Calif.). Vectashield Mounting Medium with DAPIwas purchased from Vector Laboratories, Inc. (Burlingame, Calif.). ThePolyclonal antibody, specific to the PARP cleavage site andFITC-conjugated, was acquired from Biosource (Camarillo, Calif.).CaspACE FITC-VAD-FMK marker was purchased fiom Promega (Madison, Wis.).

Synthesis of Synthetic Tea Polyphenol Analogs. Synthesis of 1, 2, 2a, 3,3a, 4, and 4a (FIG. 1)

1 was prepared according to literature procedures (Kohri, T.; Nanjo, F.;Suzuki, M.; Seto, R.; Matsumoto, N.; Yamakawa, M.; Hojo, H.; Hara, Y.;Desai, D.; Amin, S.; Conaway, C. C.; Chung, F. L. J Agric Food Chem2001, 49: 1042-8), but the synthesis of 1 will be illustrated here. Forthe synthesis of 1, commercially available (−)-EGCG was used as astarting material. Treating the (−)-EGCG with acetic anhydride andpyridine overnight yielded the desired product 1 in 82% yield (FIG. 1).The structure of 1 was confirmed by 1H and 13C NMR, LRMS and HRMS.

Mp 157.1° C.

LRMS m/z (ESI) 817 (MNa⁺)

HRMS found, 817.1544; C₃₈H₃₄O₁₉Narequires 817.1592.

¹H NMR (CDCl₃, 500 MHz) δ 7.62 (s, 2H), 7.24 (s, 2H), 6.73 (s, 1H), 6.61(s, 1H), 5.64 (br s, 1H), 5.18 (s, 1H), 3.02 (m, 2H), 2.29 (s, 3H), 2.28(s, 9H), 2.27 (s, 3H), 2.24 (s, 3H), 2.23 (s, 6H).

¹³C NMR (CDCl₃, 100 MHz) δ 168.89, 168.40, 167.59, 167.43, 166.72,166.20, 163.51, 154.71, 149.72, 149.64, 143.38, 143.29, 138.93, 135.06,134.34, 127.41, 122.34, 118.79, 109.42, 109.00, 108.06, 76.46, 67.98,25.85, 21.06, 20.75, 20.54, 20.11.

(2S*,3R*)-trans-5,7-Bis(benzyloxy)-2-[3,4,5-tris(benzyloxy)phenyl]chroman-3-ol(5)

This compound was synthesized according to literature procedures (Li, L.H.; Chan, T. H. Org. Lett. 2001, 3, 739-741).

(2S*,3R*)-trans-5,7-Bis(benzyloxy)-2-[3,4,5-tris(benzyloxy)phenyl]chroman-3-yl3-(benzyloxy)benzoate (2b)

A quantity of (COCl)₂ (0.68 mL) was added to a solution of3-(benzyloxy)benzoic acid (0.12 g, 0.53 mmol) in CH₂Cl₂ (10 mL). Themixture was refluxed for 2 hours. After which the excess (COCl)₂ and thesolvent were removed by distillation and the resulting residue was driedunder vacuum overnight. The residue was redissolved in CH₂Cl₂ (5 mL) andadded to a solution of 5 (0.20 g, 0.26 mmol) and DMAP (0.08 g, 0.64mmol) in CH₂C₂ (10 mL) at 0° C. The mixture was then stirred at roomtemperature overnight. Saturated NaHCO₃ was added. The organic layer wasseparated and the aqueous layer was extracted with ethyl acetate. Theorganic layers were combined, dried (Na₂SO₄) and evaporated. The residuewas purified by column chromatography (hexane: ethyl acetate 4:1) toafford the compound 2b as a white solid (0.22 g, 88%).

LRMS m/z (ESI) 989 (MNa⁺)

HRMS found, 989.3657; C₆₄H₅₄O₉Na requires 989.3666.

¹H NMR (CDCl₃, 500 MHz) δ 7.54-7.12 (m, 30H), 6.72 (s, 2H), 6.29 (d,J=4.5 Hz, 2H), 5.50 (q, J=7.0 Hz, 1H), 5.10 (d, J=6.5 Hz, 1H), 5.05-4.95(m, 12H), 3.04 (dd, J=16.5, 6.0 Hz, 1H), 2.89 (dd, J=16.5, 6.5 Hz, 1H).

¹³C NMR (CDCl₃, 100 MHz) δ 165.20, 158.81, 158.51, 157.53, 154.68,152.73, 138.20, 137.60, 136.76, 136.64, 136.25, 133.24, 131.14, 129.32,128.49, 128.42, 128.40, 128.29, 128.00, 127.96, 127.91, 127.80, 127.69,127.62, 127.51, 127.43, 127.38, 127.10, 122.13, 119.93, 115.29, 106.13,101.23, 94.25, 93.74, 78.38, 74.98, 71.07, 70.02, 69.81, 24.18.

(2S*,3R*)-trans-5,7-Bis(hydroxy)-2-[3,4,5-tris(hydroxy)phenyl]chroman-3-yl3-(hydroxy)benzoate (2)

Suspension of 2b (0.23 g, 0.24 mmol) in THF/MeOH (28 mL/28 mL) andPd(OH)₂ (0.19 g, 20% on carbon) was placed under an H2 atmosphere. Theresulting mixture was stirred at room temperature until tlc showed thatthe reaction was completed. Then the reaction mixture was filteredthrough cotton to remove the catalyst. After evaporation, the residuewas purified by column chromatography (ethyl acetate: CH₂Cl₂2:1) toafford the product 2 as a white solid (80 mg, 79%).

LRMS m/z (ESI) 449 (MNa⁺)

HRMS found, 449.0887; C₂₂H₁₈O₉Na requires 449.0849.

¹H NMR (CD₃OD, 500 MHz) δ 7.38-6.97 (m, 4H), 6.42 (s, 2H), 5.97 (q,J=2.5 Hz, 2H), 5.40 (q, J=6.0 Hz, 1H), 5.03 (d, J=6.0 Hz, 1H), 2.85 (dd,J=16.5, 5.0 Hz, 1H), 2.74 (dd, J=16.5, 6.0 Hz, 1H).

¹³C NMR (CD₃OD, 100 MHz) δ 165.87, 157.00, 156.52, 156.03, 154.88,145.42, 132.50, 130.91, 129.17, 120.29, 119.90, 115.45, 105.00, 98.07,95.00, 94.12, 77.78, 70.15, 22.71.

(2S*,3R*)-trans-5,7-Bis(acetyloxy)-2-[3,4,5-tris(acetyloxy)phenyl]chroman-3-yl3-acetyloxybenzoate (2a)

Suspension of 2b (0.1 g, 0.1 mmol) in THF/MeOH (12 mL/12 mL) and Pd(OH)₂(0.08 g, 20% on carbon) was placed under an H₂ atmosphere. The resultingmixture was stirred at room temperature until tlc showed that thereaction was completed. Then the reaction mixture was filtered throughcotton to remove the catalyst. The filtrate was evaporated to afford thedebenzylated compound (2) which was used immediately in the next stepwithout purification. The obtained debenzylated compound was dissolvedin pyridine (4 mL) and acetic anhydride (2 mL). The resulting mixturewas stirred at room temperature for overnight. After which, the aceticanhydride and pyridine were removed in vacuo. The resulting residue wastaken up in 20 mL of CH₂Cl₂, and the solution was washed with 5×5 mL ofH₂O and 5 mL of brine, dried over Na₂SO₄, and evaporated. The crudeproduct was purified by column chromatography (hexane: ethyl acetate1:1) to afford the compound 2a as a white powder (0.061 g, 85%).

Mp 71.6° C.

LRMS m/z (ESI) 701 (MNa⁺)

HRMS found, 701.1541 C₃₄H₃₀O₁₅Na requires 701.1482.

¹H NMR (CDCl₃, 500 MHz) δ 7.79 (d, J=8.0 Hz, 1H), 7.64 (s, 1H), 7.42 (t,J=8.0 Hz, 1H), 7.28 (s, 1H), 7.17 (s, 2H), 6.69 (s, 1H), 6.64 (s, 1H),5.46 (q, J=5.5 Hz, 1H), 5.32 (d, J=5.5 Hz, 1H), 3.02 (dd, J=17.0, 5.0Hz, 1H), 2.80 (dd, J=17.0, 6.0 Hz, 1H), 2.32 (s, 3H), 2.29 (s, 3H), 2.27(s, 3H), 2.26 (s, 9H).

¹³C NMR (CDCl₃, 100 MHz) δ 169.10, 168.81, 168.24, 167.47, 166.61,164.61, 154.00, 150.42, 149.76, 149.28, 143.47, 135.74, 134.48, 130.72,129.42, 127.18, 126.68, 122.79, 118.58, 109.84, 108.81, 107.54, 69.17,23.65, 20.96, 20.92, 20.65, 20.47, 20.10.

(2S*,3R*)-trans-5,7-Bis(benzyloxy)-2-[3,4,5-tris(benzyloxy)phenyl]chroman-3-yl4-(benzyloxy)benzoate (3b)

The title compound was prepared in a similar manner as described for 2b,using 5 (0.08 g, 0.1 mmol) and 4-(benzyloxy)benzoic acid (0.049 g, 0.22mmol) giving 3b as a white solid (0.087 g, 90%).

LRMS m/z (ESI) 989 (MNa⁺)

HRMS found, 989.3666 C₆₄H₅₄O₉Na requires 989.3666.

¹H NMR (CDCl₃, 500 MHz) δ 7.91 (d, J=9.0 Hz, 2H), 7.47-7.22 (m, 30H),6.95 (d, J=9.0 Hz, 2H), 6.74 (s, 2H), 6.31 (dd, J=5.5, 2.5 Hz, 2H), 5.52(q, J=6.5 Hz, 1H), 5.14 (d, J=6.5 Hz, 1H), 5.07-4.97 (m, 12H), 3.02 (dd,J=17.0, 5.5 Hz, 1H), 2.87 (dd, J=16.5, 7.0 Hz, 1H).

¹³C NMR (CDCl₃, 100 MHz) δ 165.08, 162.49, 158.76, 157.53, 154.65,152.69, 138.09, 137.63, 136.79, 136.70, 136.66, 136.00, 133.38, 131.61,128.55, 128.49, 128.40, 128.29, 128.09, 127.96, 127.91, 127.78, 127.69,127.62, 127.43, 127.38, 127.30, 127.09, 122.40, 114.33, 106.08, 101.30,94.22, 93.67, 78.37, 74.98, 71.05, 69.95, 69.79, 69.26, 24.01.

(2S*,3R*)-trans-5,7-Bis(hydroxy)-2-[3,4,5-tris(hydroxy)phenyl]chroman-3-yl4-(hydroxy)benzoate (3)

The title compound was prepared in a similar manner as described for 2using 3b (0.24 g, 0.25 mmol) to afford 3 as a white solid (79 mg, 75%).

LRMS m/z (ESI) 449 (MNa⁺)

HRMS found, 449.0840; C₂₂H₁₈O₉Na requires 449.0849.

¹H NMR (CD₃OD, 500 MHz) δ 7.75 (d, J=8.5 Hz, 2H), 6.77 (d, J=8.5 Hz,2H), 6.42 (s, 2H), 5.95 (dd, J=8.0, 2.5 Hz, 2H), 5.35 (q, J=6.0 Hz, 1H),5.00 (d, J=6.0 Hz, 1H), 2.85 (dd, J=16.5, 5.0 Hz, 1H), 2.71 (dd, J=16.5,6.5 Hz, 1H).

¹³C NMR (CD₃OD, 100 MHz) δ 165.88, 161.99, 156.54, 156.05, 154.95,145.41, 132.46, 131.31, 129.25, 120.61, 114.57, 104.98, 98.14, 94.91,94.04, 78.00, 69.81, 22.98.

(2S*,3R*)-trans-5,7-Bis(acetyloxy)-2-[3,4,5-tris(acetyloxy)phenyl]chroman-3-yl4-(acetyloxy)benzoate (3a)

The title compound was prepared in a similar manner as described for 2a,using 3b (0.15 g, 0.16 mmol) to afford 3a as a white solid (92.6 mg,88%).

Mp 189.4° C.

LRMS m/z (ESI) 701 (MNa⁺)

HRMS found, 701.1467; C₃₄H₃₀O₁₅Na requires 701.1482.

¹HNMR (CDCl₃, 500 MHz) δ 7.94 (d, J=9.0 Hz, 2H), 7.17 (s, 2H), 7.13 (d,J=9.0 Hz, 2H), 6.69 (d, J=2.0 Hz, 1H), 6.63 (d, J=2.0 Hz, 1H), 5.45 (q,J=6.0 Hz, 1H), 5.32 (d, J=6.0

Hz, 1H), 3.01 (dd, J=16.5, 5.0 Hz, 1H), 2.79 (dd, J=16.5, 6.0 Hz, 1H),2.31 (s, 3H), 2.29 (s, 3H), 2.27 (s, 3H), 2.26 (s, 9H).

¹³C NMR (CDCl₃, 100 MHz) δ 168.81, 168.64, 168.26, 167.49, 166.62,164.74, 154.48, 154.02, 149.76, 149.30, 143.49, 135.82, 134.50, 131.28,126.76, 121.60, 118.59, 109.90, 108.80, 107.51, 69.01, 23.64, 20.97,20.63, 20.46, 20.00.

(2S*,3R*)-trans-5,7-Bis(benzyloxy)-2-[3,4,5-tris(benzyloxy)phenyl]chroman-3-yl3,5-bis(benzyloxy)benzoate (4b)

The title compound was prepared in a similar manner as described for 2b,using 5 (0.3 g, 0.4 mmol) and 3,5-bis(benzyloxy)benzoic acid (0.27 g,0.81 mmol) giving 4b as a white solid (0.36 g, 85%).

LRMS m/z (ESI) 1095 (MNa⁺)

HRMS found, 1095.4059; C₇₁H₆₀O₁₀Na requires 1095.4084.

¹HNMR (CDCl₃, 500 MHz) δ 7.47-7.17 (m, 35H), 6.76 (s, 1H), 6.73 (s, 1H),6.30 (d, J=4.5 Hz, 2H), 5.48 (q, J=7.0 Hz, 1H), 5.08 (d, J=7.0 Hz, 1H),5.04-4.92 (m, 14H), 3.07 (dd, J=17.0, 5.5 Hz, 1H), 2.85 (dd, J=17.0, 7.0Hz, 1H).

¹³C NMR (CDCl₃, 100 MHz) δ 165.14, 159.73, 158.98, 157.67, 154.88,152.87, 137.77, 136.91, 136.82, 136.78, 136.29, 133.35, 131.86, 128.61,128.56, 128.52, 128.41, 128.15, 128.10, 128.02, 127.94, 127.81, 127.76,127.67, 127.51, 127.24, 108.52, 106.93, 106.30, 101.38, 94.43, 93.93,78.57, 75.12, 71.17, 70.26, 70.15, 70.11, 69.92, 24.58.

(2S*,3R*)-trans-5,7-Bis(hydroxy)-2-[3,4,5-tris(hydroxy)phenyl]chroman-3-yl3,5-bis(hydroxy)benzoate (4)

The title compound was prepared in a similar manner as described for 2using 4b (0.17 g, 0.16 mmol) to afford 4 as a white solid (50 mg, 71%).

LRMS m/z (ESI) 465 (MNa⁺)

HRMS found, 465.0844; C₂₂H₁₈O₉Na requires 465.0798.

¹H NMR (CD₃OD, 500 MHz) δ 6.85 (d, J=2.0 Hz, 2H), 6.44 (t, J=2.0 Hz,1H), 6.41 (s, 2H), 5.96 (s, 2H), 5.40 (dd, J=10.5, 5.0 Hz, 1H), 5.05 (d,J=5.0 Hz, 1H), 2.80 (dd, J=16.5, 5.0 Hz, 1H), 2.73 (dd, J=16.5, 5.0 Hz,1H).

¹³C NMR (CD₃OD, 100 MHz) δ 165.81, 158.10, 156.56, 156.06, 154.83,145.43, 132.44, 131.49, 129.28, 107.34, 106.86, 104.81, 97.93, 94.88,94.06, 77.60, 69.95, 22.28.

(2S*,3R*)-trans-5,7-Bis(acetyloxy)-2-[3,4,5-tris(acetyloxy)phenyl]chroman-3-yl3,5-bis(acetyloxy)benzoate (4a)

The title compound was prepared in a similar manner as described for 2ausing 4b (0.15 g, 0.14 mmol) to afford 4a as a white solid (72 mg, 70%).

Mp 105.5° C.

LRMS m/z (ESI) 759 (MNa⁺)

HRMS found, 759.1530; C₃₆H₃₂O₁₇Na requires 759.1537.

¹H NMR (CDCl₃, 500 MHz) δ 7.54 (d, J=2.0 Hz, 2H), 7.17 (s, 2H), 7.13 (t,J=2.0 Hz, 1H), 6.69 (d, J=2.0 Hz, 1H), 6.64 (d, J=2.0 Hz, 1H), 5.45 (q,J=6.0 Hz, 1H), 5.29 (d, J=6.5 Hz, 1H), 3.01 (dd, J=17.0, 5.0 Hz, 1H),2.79 (dd, J=17.0, 6.5 Hz, 1H), 2.30 (s, 6H), 2.28 (s, 3H), 2.27 (s, 3H),2.26 (s, 3H), 2.25 (s, 6H).

¹³C NMR (CDCl₃, 100 MHz) δ 168.80, 168.62, 168.21, 167.45, 166.60,163.78, 153.96, 150.78, 149.77, 149.27, 143.46, 135.55, 134.48, 131.26,120.57, 120.29, 118.56, 109.79, 108.86, 107.57, 69.48, 23.77, 20.94,20.86, 20.64, 20.45, 19.99.

As the carboxylate or —OH groups of the compound 1, 2, 3 and 4 canreadily undergo acyl exchange, one skilled in the art shall be able toreplace the acyl groups on the carboxylate groups of these compounds.

Synthesis of Synthetic Tea Polyphenol Analogs General

Starting materials and reagents, purchased from commercial suppliers,were used without further purification. Literature procedures were usedfor preparation of (2R, 3S) trans and (2R,3R)-cis-5,7-bis(benzyloxy)-2-(4-bezyloxyphenyl)chroman-3-ol, (2R, 3S)trans and (2R,3R)-cis-5,7-bis(benzyloxy)-2-[3,4-bis(benzyloxy)-phenyl]chroman-3-ol,(2R, 3R)-cis-5,7-bis(hydroxyl)-2-(4-hydroxyphenyl)chroman-3-yl3,4,5-trihydroxybenzoate (Sheng Biao Wan; Tak Hang Chan. Tetrahedron,2004, 60, 8207).

Anhydrous THF was distilled under nitrogen from sodium benzophenoneketyl. Anhydrous methylene chloride was distilled under nitrogen fromCaH₂. Anhydrous DMF was distilled under vacuum from CaH₂. Reactionflasks were flame-dried under a stream of N₂. All moisture-sensitivereactions were conducted under a nitrogen atmosphere. Flashchromatography was carried out using silica-gel 60 (70-230 mesh). Themelting points were uncorrected. ¹H-NMR and ¹³C NMR (400 MHz) spectrawere measured with TMS as an internal standard when CDCl₃ and acetone-d6were used as a solvent. High-resolution (ESI) MS spectra were recordedusing a QTOF-2 Micromass spectrometer.

(+)-(2R,3S)-5,7-Bis(benzyloxy)-2-[3,4-bis(benzyloxy)phenyl]chroman-3-yl4-benzyloxybenzoate (102)

Under an N₂ atmosphere, a solution of 4-benzyloxybenzoic acid (140 mg,0.61 mmol) was refluxed with oxally chloride (1 mL) in dry CH₂Cl₂ (10mL) and one drop of DMF for 3 h. The excess oxally chloride and solventwere removed by distillation and the residue was dried under vacuum for3 h and dissolved in CH₂Cl₂ (2 mL). This solution was added dropwise toa solution of(2R,3S)-trans-5,7-bis(benzyloxy)-2-[3,4-bis(benzyloxy)phenyl]chroman-3-ol(195 mg, 0.3 mmol) and DMAP (75 mg, 0.62 mmol) in CH₂Cl₂ (15 mL) at 0°C. The mixture was stirred at rt overnight, then saturated NaHCO₃aqueous solution was added. The organic layer was separated, and theaqueous layer was extracted with CH₂Cl₂. The organic phases werecombined, dried (MgSO₄) and evaporated. The residue was purified byflash chromatography on silica gel (EtOAc/n-hexane=1/4 v/v) to affordthe desired compound (220 mg, 85.0%). Recrystallization in EtOAc andn-hexane gave a white powder: mp 148-150° C.; [α]_(D)=+18.3 (c=1,CHCl₃); ¹H NMR (CDCl₃, 400 MHz): δ 7.88 (d, J=8.6 Hz, 2 H), 7.43-7.28(m, 25 H), 7.01 (s, 1 H), 6.93-6.88 (m, 4 H), 6.28 (s, 2H), 5.53-5.50(m, 1H), 5.14 (d, J=6.4 Hz, 1 H), 5.11 (s, 2 H), 5.07 (s, 2 H), 5.04 (S,2 H), 5.02 (s, 2 H), 5.01 (s, 2 H), 3.04 (A of ABq, J=16.8, 4.6 Hz, 1H), 2.87 (B of ABq, J=16.8, 6.3 Hz, 1 H); ¹³C NMR (CDCl₃, 400 MHz): δ165.0, 162.3, 158.6, 157.4, 154.6, 148.5, 136.9, 136.6, 136.5, 135.9,131.4, 130.9, 128.4, 128.3, 128.2, 128.1, 127.9, 127.7, 127.6, 127.5,127.3, 127.1, 126.9, 122.3, 119.5, 114.6, 114.1, 113.1, 101.1, 94.1,93.4, 78.0, 71.0, 70.9, 69.9, 69.8, 69.6, 69.1, 23.8; HRMS (ESI) calcdfor C₅₇H₄₈O₈Na (M+Na) 883.3247, found 883.3241.

(−)-(2R,3R)-5,7-Bis(benzyloxy)-2-[3,4-bis(benzyloxy)phenyl]chroman-3-yl4-benzyloxybenzoate (104)

Following the preparation procedure of 102, the esterification of (2R,3R)-cis-5,7-bis(benzyloxy)-2-[3,4-bis(benzyloxy)phenyl]chroman-3-ol with4-benzyloxybenzoic acid afforded 104 with 86% yield. mp 149-151° C.;[α]_(D)=−3.1 (c=1.5, CHCl₃); ¹H NMR (CDCl₃, 400 MHz): δ 7.92 (d, J=8.8Hz, 2 H), 7.44-7.26 (m, 25 H), 7.15 (d, J=1.6 Hz, 1 H), 6.91-6.87 (m, 4H), 6.32 (d, J=2.1 Hz, 1 H), 6.28 (d, J=2.1 Hz, 1 H), 5.63 (bs, 1 H),5.08 9s, 2 H), 5.06 (s, 1 H), 5.03-5.00 (m, 6 H), 4.92 (AB, J=11.6 Hz, 2H), 3.08 (bs, 2H); 13C NMR (CDCl3, 400 MHz): δ165.1, 162.5, 158.6,157.9, 155.6, 148.9, 148.7, 137.1, 137.0, 136.8, 136.7, 136.0, 131.8,131.1, 128.6, 128.5, 128.4, 128.3, 128.1, 127.9, 127.8, 127.6, 127.5,127.4, 127.3, 127.2, 127.1,122.5, 119.9, 114.6, 114.3, 113.5, 100.9,94.6, 93.7, HRMS (ESI) calcd for C₅₇H₄₈O₈Na (M+Na) 883.3244, found883.3241.

(+)-(2R,3S)-5,7-Bis(benzyloxy)-2-(4-benzyloxyphenyl)chroman-3-yl4-benzyloxybenzoate (110)

Following the preparation procedure of 102, the esterification of (2R,3S)-5,7-bis(benzyloxy)-2-(4-benzyloxyphenyl)chroman-3-ol with4-benzyloxybenzoic acid afforded 110 with 86% yield. mp 117-119° C.;[α]_(D)=+24.5 (c=1.2, CHCl₃); ¹H NMR (CDCl₃, 400 MHz): δ 7.86 (d, J=8.8Hz, 2 H), 7.42-7.26 (m, 22 H), 6.92 (AB, J=2.6 Hz, 4 H), 6.29 (AB, J=1.9Hz, 2 H), 5.56-5.51 (m, 1 H), 5.17 (d, J=6.5 Hz, 1 H), 5.05 (s, 2 H),4.98 (s, 4 H), 4.97 (s, 2 H), 3.10 (A of ABq, J=16.8, 5.4 Hz, 1 H), 2.88(B of ABq, J=16.8, 6.6 Hz, 1 H); ¹³C NMR (CDCl₃, 400 MHz): δ165.2,162.4, 158.8, 158.6, 157.6, 155.0, 136.8, 136.7, 136.1, 131.6, 130.3,128.6, 128.5, 128.4, 128.1, 127.9, 127.8, 127.5, 127.3, 127.1, 122.5,114.8, 114.3, 101.4, 94.3, 93.6, 78.3, 70.0, 69.9, 69.8, 69.4, 24.2;HRMS (ESI) calcd for C₅₀H₄₂O₇Na (M+Na) 777.2828, found 777.2840.

(−)-(2R,3R)-5,7-Bis(benzyloxy)-2-(4-benzyloxyphenyl)chroman-3-yl4-benzyloxybenzoate (111)

Following the preparation procedure of 102, the esterification of (2R,3R)-5,7-bis(benzyloxy)-2-(4-benzyloxyphenyl)chroman-3-ol with4-benzyloxybenzoic acid afforded 111 with 88% yield. mp 129-131° C.;[α]_(D)=−51.8 (c=3.9, CHCl₃); ¹H NMR (CDCl₃, 400 MHz): δ 7.88 (d, J=8.8Hz, 2 H), 7.43-7.27 (m, 22 H), 6.91 (s, 2 H), 6.89 (s, 2 H), 6.33 (d,J=2.0 Hz, 1 H), 6.27 (d, J=2.0 Hz, 1 H), 5.62 (bs, 1 H), 5.11 (bs, 1 H),5.05 (s, 2 H), 5.01 (s, 2 H), 4.98 (s, 4 H), 3.09 (d, J=2.9 Hz, 2 H);¹³C NMR (CDCl₃, 400 MHz): δ 165.3, 162.4, 158.6, 158.4, 157.9, 155.6,136.8, 136.7, 136.1, 131.7, 130.1, 128.6, 128.5, 128.4, 128.1, 127.9,127.8, 127.7, 127.5, 127.4, 127.3, 127.1, 122.5, 114.5, 114.3, 100.9,94.6, 93.7, 77.4, 70.0, 69.9, 69.8, 68.1, 26.0; HRMS (ESI) calcd forC₅₀H₄₂O₇Na (M+Na) 777.2828, found 777.2815.

(+)-(2R,3S)-5,7-dihydroxy-2-(3,4-dihydroxyphenyl)chroman-3-yl4-hydroxybenzoate (11)

Under an H₂ atmosphere, Pd(OH)₂/C (20%, 100 mg) was added to a solutionof 102 (200 mg, 0.23 mmol) in a solvent mixture of THF/MeOH (1:1 v/v, 20mL). The resulting reaction mixture was stirred at r.t. under H₂ for 6h, TLC showed that the reaction was completed. The reaction mixture wasfiltered to remove the catalyst. The filtrate was evaporated, and theresidue was rapidly purified by flash chromatograph on silica gel (10%MeOH/CH₂Cl₂, then 20% MeOH/CH₂Cl₂) to afford 11 (82 mg, 87% yield): mp220-222° C. (decomposed); [α]_(D)=+87.2 (c=2.0, EtOH); ¹H NMR(acetone-d₆, 400 MHz): δ 7.98 (d, J=8.8 Hz, 2 H), 7.13 (s, 1 H), 7.04(d, J=8.8 Hz, 2 H), 6.99-6.96 (m, 2 H), 6.27 (d, J=2.2 Hz, 1 H), 6.19(d, J=2.2 Hz, 1 H), 5.62 (dd, J=11.8, 6.3 Hz, 1 H), 5.34 (d, J=6.3 Hz),3.15 (A of ABq, J=16.5, 5.2 Hz, 1 H), 3.00 (B of ABq, J=16.5, 6.4 Hz, 1H); ¹³C NMR (acetone-d6, 400 MHz): δ 163.3, 160.0, 155.2, 154.5, 153.6,143.1, 129.9, 128.7, 119.6, 116.6, 113.4, 113.3, 111.8, 111.7, 96.7,93.8, 93.0, 76.2, 67.9, 21.9; HRMS (ESI) calcd for C₂₂H₁₈O₈Na (M+Na)433.0899, found 433.0909.

(−)-(2R, 3R)-5,7-dihydroxy-2-(3,4-dihydroxyphenyl)chroman-3-yl4-hydroxybenzoate (12)

Following the preparation procedure of 11, the hydrogenolysis of 104afforded 12 with 85% yield. mp 241-243° C. (decomposed); [α]_(D)=−145.2(c=0.5, EtOH), (Lit −144.4, c=1 in Me₂CO);⁴ ¹H NMR (acetone-d₆, 400MHz): δ 7.89 (d, J=8.8 Hz, 2 H), 7.19 (d, J=1.9 Hz, 1 H), 7.01 (A ofABq, J=8.2, 1.9 Hz, 1 H), 6.97 (d, J=8.8 Hz, 2 H), 6.89 (B of AB, J=8.2Hz, 1 H), 6.17 (d, J=2.3 Hz, 1 H), 6.14 (d, J=2.3 Hz, 1 H), 5.65 (m, 1H), 5.24 (bs, 1 H), 3.19 (A of ABq, J=17.4, 4.5 Hz, 1 H), 3.07 (B of AB,J=17.4, 2.0 Hz, 1 H); 13C NMR (acetone-d₆, 400 MHz): δ 164.8, 161.6,156.8, 156.4, 156.0, 144.6, 144.5, 131.5, 130.3, 121.4, 118.0, 114.9,114.6, 113.8, 97.8, 95.4, 94.7, 76.9, 68.5, 25.5; HRMS (ESI) calcd forC₂₂HI₈O₈Na (M+Na) 433.0899, found 433.0895.

(+)-(2R, 3S)-5,7-dihydroxy-2-(4-hydroxyphenyl)chroman-3-yl4-hydroxybenzoate (112)

Following the preparation procedure of 11, the hydrogenolysis of 110afforded 112 with 90% yield. mp 253-255° C. (decomposed); [α]_(D)=+45.9(c=3.5, EtOH); ¹H NMR (acetone-d₆, 400 MHz): δ 7.74 (d, J=8.7 Hz, 2 H),7.26 (d, J=8.5 Hz, 2 H), 6.81 (d, J=8.7 Hz, 2 H), 6.76 9d, J=8.5 Hz, 2H), 6.04 (d, J=2.2 Hz, 1 H), 5.95 (d, J=2.2 Hz, 1 H), 5.39 (dd, J=12.4,6.9 Hz, 1 H), 5.11 (d, J=6.9 Hz, 1 H), 3.21 (A of ABq, J=16.3, 5.3 Hz, 1H), 2.98 (B of ABq, J=16.3, 7.0 Hz, 1 H); 13C NMR (acetone-d₆, 400 MHz):δ 163.7, 160.6, 156.0, 155.9, 155.1, 154.3, 130.4, 128.3, 126.8, 120.1,113.9, 97.3, 94.4, 93.5, 77.0, 68.5, 23.0; HRMS (ESI) calcd forC₂₂H₁₈O₇Na (M+Na) 417.0950, found 417.0946.

(−)-(2R, 3R)-5,7-dihydroxy-2-(4-hydroxyphenyl)chroman-3-yl4-hydroxybenzoate (10)

Following the preparation procedure of 11, the hydrogenolysis of 111afforded 10 with 89% yield. mp 214-216° C. (decomposed); [α]_(D)=−116.1(c=2.0, EtOH); ¹H NMR (acetone-d₆, 400 MHz): δ 7.94 (d, J=8.6 Hz, 2 H),7.56 (d, J=8.6 Hz, 2 H), 7.01 (d, J=6.8 Hz, 2 H), 6.95 (d, J=6.8 Hz, 2H), 6.22 (d, J=2.2 Hz, 1 H), 6.20 (d, J=2.2 Hz, 1 H), 5.73 (bs, 1 H),5.37 (bs, 1 H), 3.26 (A of ABq, J=17.4, 4.5 Hz, 1 H), 3.14 (B of ABq,J=17.4, 2.2 Hz, 1 H); 13C NMR (acetone-d₆, 400 MHz): δ 164.5, 161.3,156.6, 156.5, 156.1, 155.8, 131.2, 129.3, 127.5, 121.1, 114.7, 114.4,97.5, 95.2, 94.5, 76.7, 68.2, 25.2; HRMS (ESI) calcd for C₂₂H₁₈O₇Na(M+Na) 417.0950, found 417.0946.

(+)-(2R,3S)-5,7-dihydroxy-2-(3,4-dihydroxyphenyl)chroman-3-yl4-hydroxybenzoate pentaacetate (22*)

Under an N₂ atmosphere, to a solution of (+)-(2R,3S)-5,7-dihydroxy-2-(3,4-dihydroxyphenyl)chroman-3-yl 4-hydroxybenzoate11 (20 mg, 0.048 mmol) in pyridine (1 ml), acetic anhydride (0.2 ml) wasadded dropwise at 0° C. The reaction mixture was stirred at rtovernight. The excess pyridine was distilled under vacuum. The residuewas purified by flash chromatograph on silica gel (EtOAc/n-hexane, 1/1in v/v) to afford 22* (34 mg, 95% yield). mp: mp 149-151° C.;[α]_(D)=+42.5 (c=1.2, CHCl₃); ¹H NMR (CDCl₃, 400 MHz): δ 7.96 9d, J=8.7Hz, 2 H), 7.31 (A of ABq, J=8.4, 1.8 Hz, 1 H), 7.26 (d, J=1.8 Hz, 1 H),7.21 (B of AB, J=8.4 Hz, 1 H), 7.15 (d, J=8.7 Hz, 2 H), 6.70 (d, J=2.2Hz, 1 H), 6.63 (d, J=2.2 Hz, 1 H), 5.51 (dd, J=11.2, 6.0 Hz, 1H), 5.33(d, J=6.0 Hz, 1 H), 3.04 (A of ABq, J=16.8, 5.0 Hz, 1 H), 2.83 (B ofABq, J=16.8, 6.0 Hz, 1 H), 2.31 (s, 3 H), 2.29 (s, 3 H), 2.28 (s, 3 H),2.27 (s, 6 H); ¹³C NMR (CDCl₃, 400 MHz): δ 168.8, 168.7, 168.3, 167.9,164.8, 154.5, 154.3, 149.8, 149.4, 142.2, 142.1, 136.1, 131.3, 126.9,124.3, 123.7, 122.0, 121.6, 121.3, 110.0, 108.7, 107.6, 77.7, 69.1,23.9, 21.0, 20.7, 20.5; HRMS (ESI) calcd for C₃₂H₂₈O₁₃Na (M+Na)643.1428; found 643.1437.

(−)-(2R,3R)-5,7-dihydroxy-2-(3,4-dihydroxyphenyl)chroman-3-yl4-hydroxybenzoate pentaacetate (23*)

Following the preparation procedure of 22*, the acetylation of 12afforded 23* with 96% yield. mp 91-93° C.; [α]_(D)=−26.5 (c=0.5, CHCl₃);¹H NMR (CDCl₃, 400 MHz): δ 7.89 (d, J=8.7 Hz, 2 H), 7.37 (d, J=1.9 Hz, 1H), 7.34 (A of ABq, J=8.4, 1.9 Hz, 1 H), 7.21 (B of AB, J=8.4 Hz, 1 H),7.10 (d, J=8.7 Hz, 2 H), 6.74 (d, J=2.2 Hz, 1 H), 6.59 (d, J=2.2 Hz, 1H), 5.64 (bs, 1 H), 5.22 (bs, 1 H), 3.11 (A of ABq, J=18.0, 4.5 Hz, 1H), 3.04 (B of ABq, J=18.0, 2.4 Hz, 1 H), 2.29 (s, 3 H), 2.28 (s, 6 H),2.26 (s, 3H), 2.25 (s, 3 H); ¹³C NMR (CDCl₃, 400 MHz): δ 168.8, 168.6,168.3, 167.9, 167.8, 164.9, 154.9, 154.4, 149.7, 142.0, 141.8, 135.7,131.3, 126.9, 124.2, 123.4, 121.7, 121.5, 109.5, 108.8, 108.0, 67.5,26.0, 21.0, 20.7, 20.5; HRMS (ESI) calcd for C₃₂H₂₈O₁₃Na (M+Na)643.1428; found 643.1420.

(+)-(2R,3S)-5,7-dihydroxy-2-(3,4-dihydroxyphenyl)chroman-3-yl3,4,5-trihydroxybenzoate heptaacetate (24*)

Following the preparation procedure of 22*, the acetylation of (+)-(2R,3S)-5,7-dihydroxy-2-(3,4-dihydroxyphenyl)chroman-3-yl3,4,5-trihydroxybenzoate (Sheng Biao Wan; Di Chen; Q. Ping Dou and TakHang Chan. Bioorganic & Medicinal Chemistry, 2004, 12, 3521) afforded24* with 95% yield. mp 140-142° C.; [α]_(D)=+35.3 (c=3.0, CHCl₃); ¹H NMR(CDCl₃, 400 MHz): δ 7.67 (s, 2 H), 7.28-7.26 (m, 2 H), 7.20 (d, J=8.3Hz, 1 H), 6.68 (d, J=2.2 Hz, 1 H), 6.62 (d, J=2.2 Hz, 1 H), 5.47 (dd,J=11.8, 6.2 Hz, 1 H), 5.27 (d, J=6.2 Hz, 1 H), 3.02 (A of ABq, J=16.8,5.2 Hz, 1 H), 2.81 (B of ABq, J=16.8, 6.6 Hz, 1 H), 2.29 (m, 9 H), 2.27(s, 3 H), 2.26 (s, 3 H), 2.25 (s, 6 H); ¹³C NMR (CDCl₃, 400 MHz): δ168.8, 168.2, 167.8, 167.5, 166.2, 163.3, 154.2, 149.8, 149.3, 143.3,142.1, 138.9, 135.7, 127.5, 124.2, 123.7, 122.2, 121.6, 109.9, 108.8,107.6, 77.5, 69.7, 24.0, 21.0, 20.7, 20.5, 20.0; HRMS (ESI) calcd forC₃₆H₃₂O₁₇Na (M+Na) 759.1537; found 759.1552.

(−)-(2R,3R)-5,7-dihydroxy-2-(3,4-dihydroxyphenyl)chroman-3-yl3,4,5-trihydroxybenzoate heptaacetate (25*)

Following the preparation procedure of 22*, the acetylation of (−)-(2R,3R)-5,7-dihydroxy-2-(3,4-dihydroxyphenyl)chroman-3-yl3,4,5-trihydroxybenzoate (Sheng Biao Wan; Di Chen; Q. Ping Dou and TakHang Chan. Bioorganic & Medicinal Chemistry, 2004, 12, 3521) afforded25* with 93% yield. mp 105-107° C.; [α]_(D)=−14.5 (c=1.2, CHCl₃); ¹H NMR(CDCl₃, 400 MHz): δ 7.62 (s, 2 H), 7.33 (d, J=1.2 Hz, 1 H), 7.31 (A ofAB, J=8.4 Hz, 1 H), 7.20 (B of AB, J=8.4 Hz, 1 H), 6.73 (d, J=2.0 Hz, 1H), 6.60 (d, J=2.0 Hz, 1 H), 5.63 (bs, 1 H), 5.20 (bs. 1 H), 3.10 (A ofABq, J=18.0, 4.5 Hz, 1 H), 3.00 (B of AB, J=18.0 Hz, 1 H), 2.28-2.27 (m,15 H), 2.25 (s, 3 H), 2.23 (s, 3 H); ¹³C NMR (CDCl₃, 400 MHz): δ 168.8,168.3, 167.9, 167.4, 166.2, 163.5, 154.9, 149.7, 143.3, 142.0, 141.9,138.9, 135.3, 127.4, 124.3, 123.5, 122.2, 121.8, 109.4, 108.9, 108.0,68.1, 25.9, 21.0, 20.7, 20.5, 20.1; HRMS (ESI) calcd for C₃₆H₃₂O₁₇Na(M+Na) 759.1537; found 759.1571.

(+)-(2R, 3S)-5,7-dihydroxy-2-(4-hydroxyphenyl)chroman-3-yl4-hydroxybenzoate tetraacetate (114)

Following the preparation procedure of 22*, the acetylation of 112afforded 114 with 91% yield. mp 167-169° C.; [α]_(D)=+3.2 (c=1.0,CHCl₃); ¹H NMR (CDCl₃, 400 MHz): δ 7.94 (d, J=8.7 Hz, 2 H), 7.42 (d,J=8.5 Hz, 2 H), 7.14 (d, J=8.7 Hz, 2 H), 7.09 (d, J=8.5 Hz, 2 H), 6.69(d, J=2.2 Hz, 2 H), 6.61 (d, J=2.2 Hz, 2 H), 5.53 (dd, J=11.5, 6.3 Hz, 1H), 5.31 (d, J=6.3 Hz, 2 H), 3.01 (A of ABq, J=16.7, 5.1 Hz, 1 H), 2.81(B of ABq, J=16.7, 6.3 Hz, 1 H), 2.30 (s, 3 H), 2.28 (s, 3 H), 2.27 (s,3 H), 2.25 (s, 3 H); ¹³C NMR (CDCl₃, 400 MHz): δ 169.1, 168.8, 168.7,168.3, 164.7, 154.6, 154.5, 150.7, 149.8, 149.4, 134.8, 131.2, 127.5,127.0, 121.8, 121.6, 110.2, 108.6, 107.6, 78.1, 69.1, 24.1, 21.0, 20.7;HRMS (ESI) calcd for C₃₀H₂₇O₁₁ (M+H) 563.1553; found 563.1567; and calcdfor C₃₀H₂₆O₁₁Na (M+Na) 585.1373; found 585.1387.

(−)-(2R, 3R)-5,7-dihydroxy-2-(4-hydroxyphenyl)chroman-3-yl4-hydroxybenzoate tetraacetate (21*)

Following the preparation procedure of 22*, the acetylation of 10afforded 21* with 89% yield. mp 144-145° C.; [α]_(D)=−30.7 (c=2.5,CHCl₃); ¹H NMR (CDCl₃, 400 MHz): δ 7.90 (d, J=8.7 Hz, 2 H), 7.49 (d,J=8.5 Hz, 2 H), 7.10 (d, J=8.5 Hz, 2 H), 7.08 (d, J=8.7 Hz, 2 H), 6.74(d, J=2.2 Hz, 2 H), 6.59 (d, J=2.2 Hz, 2 H), 5.64 (bs, 1 H), 5.22 (s, 1H), 3.12 (A of ABq, J=17.7, 4.4 Hz, 1 H), 3.02 (B of ABq, J=17.7, 1.8Hz, 1 H), 2.28 (s, 6 H), 2.27 (s, 3 H), 2.25 (s, 3 H); ¹³C NMR (CDCl₃,400 MHz): δ 169.1, 168.9, 168.7, 168.4, 164.8, 155.2, 154.4, 150.5,149.7, 134.5, 131.2, 127.4, 126.9, 121.6, 109.7, 108.7, 108.0, 67.7,26.2, 21.0, 20.7; HRMS (ESI) calcd for C₃₀H₂₆O₁₁Na (M+Na) 585.1373;found 585.1371.

(+)-(2R, 3S)-5,7-dihydroxy-2-(4-hydroxyphenyl)chroman-3-yl3,4,5-trihydroxybenzoate hexaacetate (16*)

Following the preparation procedure of 22*, the acetylation of (+)-(2R,3S)-5,7-dihydroxy-2-(4-hydroxyphenyl)chroman-3-yl3,4,5-trihydroxybenzoate (Sheng Biao Wan; Tak Hang Chan. Tetrahedron,2004, 60, 8207) afforded 16* with 93% yield. mp 96-98° C.; [α]_(D)=+17.7(c=1.0, CHCl₃); ¹H NMR (CDCl₃, 400 MHz): δ 7.66 (s, 2 H), 7.41 (d, J=8.5Hz, 2 H), 7.10 (d, J=8.5 Hz, 2 H), 6.68 (d, J=2.2 Hz, 2 H), 6.62 (d,J=2.2 Hz, 2 H), 5.51 (dd, J=12.2, 6.7 Hz, 1 H), 5.26 (d, J=6.7 Hz, 1 H),3.02 (A of ABq, J=16.7, 5.3 Hz, 1 H), 2.81 (B of ABq, J=16.7, 6.6 Hz, 1H), 2.30 (s, 6 H), 2.29 (s, 3 H), 2.28 (s, 3 H), 2.27 (s, 6 H); ¹³C NMR(CDCl₃, 400 MHz): δ 169.1, 168.8, 168.3, 167.5, 166.2, 163.3, 154.5,150.7, 149.9, 149.4, 143.4, 138.9, 134.5, 127.5, 122.2, 121.8, 110.1,108.7, 107.7, 78.0, 69.8, 24.3, 21.0, 20.7, 20.5, 20.1; HRMS (ESI) calcdfor C₃₄H₃₀O₁₅Na (M+Na) 701.1482; found 701.1490.

(−)-(2R, 3R)-5,7-dihydroxy-2-(4-hydroxyphenyl)chroman-3-yl3,4,5-trihydroxybenzoate hexaacetate (19*)

Following the preparation procedure of 22*, the acetylation of (−)-(2R,3R)-5,7-dihydroxy-2-(4-hydroxyphenyl)chroman-3-yl3,4,5-trihydroxybenzoate (Sheng Biao Wan; Tak Hang Chan. Tetrahedron,2004, 60, 8207) afforded 19* with 91% yield. mp 152-154° C.;[α]_(D)=−35.5 (c=2.5, CHCl₃); ¹H NMR (CDCl₃, 400 MHz): δ 7.62 (s, 2 H),7.46 (d, J=8.5 Hz, 2 H), 7.11 (d, J=8.5 Hz, 2 H), 6.73 (d, J=2.1 Hz, 1H), 6.60 (d, J=2.1 Hz, 1 H), 5.60 (bs, 1 H), 5.21 (s, 1 H), 3.10 (A ofABq, J=17.8, 4.5 Hz, 1 H), 3.00 (B of ABq, J=17.8, 1.8 Hz, 1 H),2.28-2.26 (m, 18 H); ¹³C NMR (CDCl₃, 400 MHz): δ 169.1, 168.8, 168.3,167.4, 166.2, 163.5, 155.1, 150.6, 149.7, 143.3, 138.8, 134.2, 127.5,127.4, 122.2, 121.7, 109.5, 108.8, 108.0, 77.2, 68.3, 26.0, 21.0, 20.7,20.5, 20.1; HRMS (ESI) calcd for C₃₄H₃₀O₁₅Na (M+Na) 701.1482; found701.1452.

(E)-3-[2,4-Bis(benzyloxy)-6-hydroxyphenyl]-1-phenyl-propene (#a)

Following the procedure in the literature (Li, L.; Chan, T. H. Org.Lett. 2001, 5, 739), cinnamyl alcohol was reacted with3,5-dibenzyloxyphenol to yield (#a) as a white solid (62% yield); mp76-78° C.; ¹H NMR (CDCl₃, 400 MHz) δ 7.40-7.24 (m, 15 H), 6.48(A of AB,J=15.9 Hz, 1 H), 6.35 (B of ABt, J=15.9, 5.5 HZ, 1 H), 6.27 (d, J=2.1Hz, 1 H), 6.16 (d, J=2.1 Hz, 1H), 5.07 (s, 1H), 5.02 (s, 2 H), 4.99 (s,2 H), 3.59-3.57 (d, J=5.5 Hz, 2 H); ¹³C NMR (CDCl₃, 400 MHz) δ 158.5,157.6, 155.4, 137.0, 136.8, 136.6, 130.2, 128.3, 128.2, 128.1, 128.0,127.8, 127.6, 127.3, 127.0, 126.8, 125.8, 106.6, 94.8, 93.4, 70.1, 69.9,26.2; HRMS (ESI) calcd for C₂₉H₂₆O₃Na (M+Na) 445.1780, found 445.1793.

(+)-(1S,2S)-3-[2,4-Bis(benzyloxy)-6-hydroxyphenyl]-1-phenylpropane-1,2-diol((+)-#b)

Following the procedure in the literature (Li, L.; Chan, T. H. Org.Lett. 2001, 5, 739), but with (#a) as starting material and AD-mix-α asdihydroxylation regent, (+)-#b was obtained (47% yield) as a whitesolid; mp 121-123° C.; [α]_(D)=+4.7 (c=0.6, CHCl₃); ¹H NMR (CDCl₃, 400MHz) δ 7.38-7.19 (m, 13 H), 7.10 (m, 2 H), 6.23 (d, J=2.3 Hz, 1 H), 6.17(d, J=2.3 Hz, 1 H), 4.94 (s, 2 H), 4.82 (AB, J=11.7 Hz, 2 H), 4.47 (d,J=6.6 Hz, 1 H), 3.97 (m, 1 H), 2.88 (A of ABt, J=14.6, 3.6 Hz, 1 H),2.72 (b of ABt, J=14.6, 8.6 Hz, 1 H); ¹³C NMR (CDCl₃, 400 MHz): δ 158.8,157.6, 157.0, 140.0, 136.6, 136.5, 128.3, 128.2, 128.1, 128.0, 127.7,127.4, 127.3, 126.8, 126.5, 105.9, 95.6, 93.2, 76.6, 69.8, 69.7, 26.2;HRMS (ESI) calcd for C₂₉H₂₈O₅Na (M+Na) 479.1834, found 479.1841.

(+)-(2S, 3S)-cis-5,7-Bis(benzyloxy)-2-phenylchroman-3-ol ((+)-#c)

Following the procedure in the literature (Li, L.; Chan, T. H. Org.Lett. 2001, 5, 739), but with (+)-#b as starting material, (+)-#c wasobtained (47% yield) as a white solid; mp 60-62° C.; [α]_(D)=+0.9(c=1.0, ethyl acetate); ¹H NMR (CDCl₃, 400 MHz) δ 7.49-7.30 (m, 15 H),6.29 d, J=2.2 Hz, 1 H), 6.27 (d, J=2.2 Hz, 1 H), 5.02 (bs, 1 H), 4.99(s, 4 H), 4.25 (bs, 1 H), 3.03 (A of ABt, J=17.2, 1.4 Hz, 1 H), 2.96 (Bof ABt, J=17.2, 4.2 Hz, 1 H); ¹³C NMR (CDCl₃, 400 MHz): δ 158.7, 158.2,155.2, 138.1, 136.9, 136.8, 128.5, 128.4, 128.0, 127.9, 127.8, 127.4,127.1, 126.2, 100.9, 94.6, 94.0, 78.6, 70.0, 69.8, 66.2, 28.2; HRMS(ESI) calcd for C₂₉H₂₆O₄Na (M+Na) 461.1729, found 461.1741.

(+)-(2S, 3S)-5,7-dihydroxy-2-phenylchroman-3-yl 3,4,5-trihydroxybenzoate((+)-15)

Following the procedure in the literature (Li, L.; Chan, T. H. Org.Lett. 2001, 5, 739), but with (+)-#c as starting material, compound(+)-15 was obtained (90% yield): mp 258-260° C. (decomposed);[α]_(D)=+13.9 (c=3.5, ethanol); ¹H NMR (acetone-d₆, 400 MHz): δ7.73-7.71 (m, 2 H), 7.49-7.45 (m, 2 H), 7.41-7.38 (m. 1 H), 7.17 (s, 2H), 6.24 (d, J=2.2 Hz, 1 H), 6.23 (d, J=2.2 Hz, 1 H), 5.76 (bs, 1 H),5.44 (bs, 1 H), 3.27 (A of ABt, J=17.4, 4.5 Hz, 1 H), 3.14 (B of ABt,J=17.4, 1.4 Hz, 1 H); ¹³C NMR (acetone-d₆, 400 MHz): δ 164.7, 156.6,156.3, 155.7, 144.7, 138.5, 137.6, 127.6, 127.3, 126.3, 120.4, 108.7,97.7, 95.4, 94.6, 77.0, 68.1, 25.3; HRMS (ESI) calcd for C₂₂H₁₈O₈Na(M+Na) 433.0899, found 433.0904.

2-Benzyl-3,5-bis(benzyloxy)phenol (#d)

Ethanethiol (10 g, 216 mmol) was added dropwise to a stirred suspensionof sodium hydride (60% dispersion in mineral oil, 2.4 g, 100 mmol) indry DMF (120 mL) at 0° C. After 1 h, 1,3,5-tribenzyloxybezene (24 g, 60mmol) was added in 10 batches and the mixture was heated to 150° C. for1.5 h. After the reaction was cooled, water (500 mL) was added and themixture was extracted with EtOAc. The combined organic layers were dried(MgSO₄) and evaporated. The residue was purified by flash chromatographyon silica gel (benzene) to give 3,5-dibenzyloxyphenol as white solid(11%) after recrystallization from carbon tetrachloride and product #das white solid (56%) after recrystallization from EtOAc and hexane.Compound #d was identified by mp 107-109° C.; ¹H NMR (CDCl₃, 400 MHz) δ7.36-7.23 (m, 15 H), 6.25 (d, J=2.2 Hz, 1 H), 6.07 (d, J=2.2 Hz, 1 H),4.96 (s, 2 H), 4.92 (s, 2 H), 3.99 (s, 2 H); ¹³C NMR (CDCl₃, 400 MHz) δ158.4, 157.9, 154.9, 140.7, 136.7, 136.6, 128.4, 128.2, 128.1, 127.8,127.5, 127.3, 127.0, 125.6, 108.5, 94.7, 93.3, 70.2, 69.9, 28.3; HRMS(ESI) calcd for C₂₇H₂₄O₃Na (M+Na) 419.1623, found 419.1645.

(E)-3-[2,4-Bis(benzyloxy)-5-benzyl-6-hydroxyphenyl]-1-[3,4-bis(beznyloxy)phenyl]-propene(#e)

At rt under an N₂ atmosphere, 25% H₂SO₄/SiO₂ (1.6 g, 4 mmol) was addedin one batch to the stirred mixture of 2-benzyl-3,5-bis(benzyloxy)phenol(3.96 g, 10 mmol) and (E)-3,4-bis(benzyloxy)cinnamyl alcohol (3.46 g, 10mmol) in dry CH₂Cl₂ (80 mL). The resulting mixture was stirred at rtovernight. After filtration and evaporation, the residue was purified bycolumn chromatography on silica gel (EtOAc/n-hexane=1/7 v/v) andrecrystalized from EtOAc and n-hexane to give a white solid, (3.4 g,46.0% yield): mp 93-95° C.; ¹H NMR (CDCl₃, 400 MHz) δ 7.41-7.22 (m, 30H), 6.92 (s, 1 H), 6.79 (s, 1 H), 6.78 (d, J=4.0 Hz, 1 H), 6.35 (A ofAB, J=15.8 Hz, 1 H), 6.26 (s, 1 H), 6.13-6.07 (B of ABt, J=15.8, 5.0 Hz,1 H), 5.08 (s, 2 H), 5.07 (s, 2 H), 4.99 (s, 4 H), 4.02 (s, 2 H), 3.55(d, J=5.0 Hz, 2 H); ¹³C NMR (CDCl₃, 400 MHz) δ 156.1, 155.6, 154.0,148.9, 148.2, 141.1, 137.2, 137.1, 131.0, 130.1, 128.5, 128.4, 128.2,127.8, 127.7, 127.6, 127.3, 127.2, 127.1, 126.5, 125.7, 119.7, 114.9,112.4, 109.6, 107.3, 91.4, 71.1, 70.5, 70.3, 28.8, 26.6; HRMS (ESI):calcd for C₅₀H₄₄O₅Na (M+Na) 747.3086, found 747.3096.

(−)-(1R,2R)-3-[2,4-Bis(benzyloxy)-5-benzyl-6-hydroxy-phenyl]-1-[3,4-bis(benzyloxy)phenyl]propane-1,2-diol((−)-#f)

The propene (#e) (3.4 g, 4.6 mmol) was dissolved in dry DMF (30 mL), andto this solution imidazole (1.03 g, 15.2 mmol) and TBSC1 (1.2 g, 7.8mmol) were added successively. The resulting mixture was stirred at rtfor 3 days, and then saturated Na₂CO₃ solution was added to quench thereaction. The mixture was extracted with EtOAc. The organic layers werecombined, dried (MgSO₄), and evaporated. The residue was purified byflash chromatograph on silica gel (n-hexane and EtOAc=9/1 v/v) to afford[3,5-bis(benzyloxy)-6-benzyl-2-[3-[3,4-bis(benzyloxy)phenyl]allyl]phenoxy]-tert-butyldimethylsilane. This material wasused in next step without further purification.

AD-mix-β (13.0 g) and methanesulfonamide (0.87 g) were dissolved in asolvent mixture of t-BuOH (50 mL) and H₂O (50 mL). The resulting mixturewas stirred at rt for 5 min, then the mixture was cooled to 0° C. and asolution of[3,5-bis(benzyloxy)-6-benzyl-2-[3-[3,4-bis(benzyloxy)phenyl]-allyl]phenoxy]-tert-butyldimethylsilanein dichloromethane (50 mL) was added. After the mixture had been stirredovernight, two more batches of AD-mix-β (13.0 g each) andmethanesulfonamide (0.87 g each) were added in each 24 h intervals.After another 24 h of stirring at 0° C., TLC showed that the reactionwas completed. Then a 10% Na₂S₂O₃ solution was added to quench thereaction. The mixture was extracted with EtOAc. The organic phases werecombined, dried (MgSO₄) and evaporated. The residue was purified byflash chromatograph on silica gel (n-hexane and EtOAc=4/1 v/v) to afford[3,5-bis(benzyloxy)-6-benzyl-2-[3-[3,4-bis(benzyloxy)phenyl]-1,2-dihydroxyl-propyl]phenoxy]-tert-butyldimethylsilane.The resulting compound was dissolved in THF (75 mL), and TBAF (10 mL, 1M in THF) was added. The resulting mixture was stirred at rt for 4 h,and saturated NaHCO₃ solution was added. The mixture was extracted withEtOAc, and the organic layers were combined, dried (MgSO4) andevaporated. The residue was purified by flash chromatography on silicagel (EtOAc/hexane=1/2 v/v) and then recrystallized from EtOAc and hexaneto give a white solid (2.4 g, 67% yield) (−)-#f: mp 157-159° C.;[α]_(D)=−5.5 (c=1.1, CHCl₃); ¹H NMR (CDCl₃, 400 MHz) δ 7.41-7.09 (m, 25H), 6.91 (d, J=1.6 Hz, 1 H), 6.77 (m, 2 H), 6.17 (s, 1 H), 5.06 (s, 4H), 4.98 (s, 2 H), 4.82 (AB, J=11.9 Hz, 2 H), 4.42 (d, J=6.6 Hz, 1 H),4.05 (s, 2 H), 3.91 (b, 1 H), 2.93 (A of ABt, J=14.5, 3.2 Hz, 1 H), 2.72(B of ABt, 14.5, 8.5 Hz, 1 H); ¹³C NMR (CDCl₃, 400 MHz) δ 156.4, 155.6,155.3, 148.9, 148.8, 142.1, 137.2, 137.1, 137.0, 133.5, 128.6, 128.5,128.4, 128.0, 127.8, 127.7, 127.6, 127.3, 127.2, 127.1, 126.7, 125.3,119.9, 114.5, 113.6, 112.1, 111.2, 106.7, 90.9, 77.2, 76.7, 71.1, 70.9,70.3, 70.2, 29.1, 26.8; HRMS (ESI) calcd for C₅₀H₄₆O₇Na (M+Na) 781.3141,found 781.3110.

(−)-(2S,3R)-trans-5,7-Bis(benzyloxy)-8-benzyl-2-[3,4-bis-(benzyloxy)phenyl]chroman-3-ol((−)-#g)

To a suspension of (−)-#f (2.4 g, 3.1 mmol) in 1,2-dichloroethane (50mL) was added triethyl orthoformate (1 mL), followed by PPTS (450 mg,1.8 mmol). The mixture was stirred at rt for 20 min until the soliddissolved. The mixture was then heated to 55° C. for 5 h until TLCshowed the reaction had been completed. After evaporation of thesolvent, the residue was dissolved in DME (30 mL) and MeOH (30 ml),K₂CO₃ (450 mg) was added. The mixture was stirred at rt overnight. Afterevaporation of the solvent, the residue was purified by flashchromatography on silica gel (EtOAc/hexane, 1/3 v/v) to afford thedesired product as white solid (1.8 g, 77% yield): mp 145-146° C.;[α]_(D)=−20.1 (c=1.3, CHCl₃); ¹H NMR (CDCl₃, 400 MHz) δ 7.42-7.15 (m, 25H), 6.93 (d, J=1.6 Hz, 1 H), 6.88 (A of AB, J=8.3 Hz, 1 H), 6.82 (B ofABt, J=8.3, 1.6 Hz, 1 H), 6.24 (s, 1 H), 5.13 (s, 2 H), 5.02 (s, 2 H),5.00 (s, 2 H), 4.98 (s, 2 H), 4.63 (d, J=8.1 Hz, 1 H), 4.04 (AB, J=14.2Hz, 2 H), 3.86 (m, 1 H), 3.10 (A of ABt, J=5.6, 16.4 Hz, 1 H), 2.67 (Bof ABt, J=9.0, 16.4 Hz, 1 H); ¹³C NMR (CDCl₃, 400 MHz) δ 155.8, 155.5,152.9, 148.9, 148.8, 142.2, 137.2, 137.1, 137.0, 136.9, 131.2, 128.7,128.5, 128.4, 128.3, 127.9, 127.8, 127.7, 127.4, 127.2, 127.1, 127.0,125.2, 120.2, 114.5, 113.2, 110.2, 102.4, 91.1, 81.2, 71.1, 70.9, 70.4,69.9, 68.2, 28.6, 27.6. HRMS (ESI) calcd for C₅₀H₄₄O₆Na (M+Na) 763.3036,found 763.3032.

(+)-(2R)-5,7-Bis(benzyloxy)-8-benzyl-2-[3,4-bis(benzyl-oxy)-phenyl]chroman-3-one((−)-#h))

Dess-Martin periodinane (6.3 mL, 15% g/mL in CH₂Cl₂, 2.2 mmol) was addedin one batch to a stirred solution of (−)-#g (900 mg, 1.2 mmol) inCH₂Cl₂ (30 mL) under an N₂ atmosphere. The mixture was stirred at rt forabout 2 h till TLC showed the absence of starting marterial.Subsequently, saturated NaHCO₃ solution (15 mL) and 10% Na₂S₂O₃ aqueoussolution (15 mL) were added to quench the reaction. The organic layerwas separated, and the aqueous layer was extracted with CH₂Cl₂. Thecombined organic phases were dried (MgSO₄) and evaporated. The residuewas purified by flash chromatography on silica gel (benzene) and thenrecrystallized in CHCl₃ and ether to afford the desired compound (770mg, 86%): mp 143-145° C., [α]_(D)=−17.1 (c=1.1, CHCl₃); ¹H NMR (CDCl₃,400 MHz): δ 7.43-7.07 (m, 25 H), 6.89 (s, 1 H), 6.84 (AB, J=8.5 Hz, 2H), 6.33 (s, 1 H), 5.13-5.04 (m, 6 H), 5.02 (s, 1 H), 4.99 (s, 2 H),4.05 (AB J=14.2 Hz, 2 H), 3.67 (AB, J=20.8 Hz, 2 H); ¹³C NMR (CDCl₃, 400MHz) δ 205.8, 156.5, 154.8, 152.5, 149.0, 148.7, 141.7, 137.1, 136.9,136.8, 136.5, 128.6, 128.5, 128.4, 128.1, 128.0, 127.8, 127.7, 127.3,127.1, 125.4, 120.0, 114.5, 113.2, 111.9, 102.4, 92.6, 82.9, 71.0, 70.5,70.2, 34.0, 28.8; HRMS (ESI) calcd for C₅₀H₄₂O₆Na (M+Na) 761.2879, found761.2843.

(+)-(2S,3S)-cis-5,7-Bis(benzyloxy)-8-benzyl-2-[3,4-bis-(benzyloxy)phenyl]chroman-3-ol((+)-#i)

Under an N₂ atmosphere, the ketone (−)-#h (700 mg, 0.95 mmol) wasdissolved in dry THF (15 mL), and the solution was cooled to −78° C.Then L-selectride (1.5 mL, 1 M solution in THF, 1.5 mmol) was addeddropwise. The resulting solution was stirred at −78° C. for 8 h. WhenTLC showed the reaction was completed, saturated NaHCO₃ aqueous solution(10 mL) was added to quench the reaction. The organic layer wasseparated and the aqueous layer was extracted with EtOAc. The combinedorganic phases were dried (MgSO₄) and evaporated. The residue waspurified by flash chromatography on silca gel (5% EtOAC/benzene) andthen recrystallized with ethanol and EtOAC to afford the desired product(630 mg, 90%) as a white solid: mp 129-131° C., [α]_(D)=+5.3 (c=1.2,CHCl₃); ¹H NMR (CDCl₃, 400 MHz): δ 7.44-7.07 (m, 25 H), 7.05 (s, 1 H),6.93 (AB, J=8.4 Hz, 2 H), 6.26 (s, 1 H), 5.15 (s, 2 H), 5.02 (s, 2 H),5.00 (s, 4 H), 4.92 (bs, 1 H), 4.20 (bs, 1 H), 4.10 (AB, J=14.5 Hz, 2H), 3.07 (A of AB, J=17.2 Hz, 1 H), 2.92 (B of ABt, J=17.2, 4.2 Hz, 1H); ¹³C NMR (CDCl₃, 400 MHz): δ 156.2, 155.9, 152.8, 148.9, 148.5,142.1, 137.2, 137.1, 137.0, 131.6, 128.5, 128.4, 128.0, 127.8, 127.7,127.4, 127.2, 125.2, 118.9, 114.9, 112.9, 110.2, 101.1, 91.4, 78.0,71.2, 71.0, 70.5, 70.0, 66.1, 28.6, 28.2; HRMS (ESI) calcd forC₅₀H₄₄O₆Na (M+Na) 763.3036 found 763.3024.

(+)-(2S,3S)-5,7-Bis(benzyloxy)-8-benzyl-2-[3,4-bis-(benzyloxy)-phenyl]chroman-3-yl3,4,5-tris(benzyloxy)-benzoate ((+)-#j).

Under an N₂ atmosphere, a solution of 3,4,5-tris(benzyloxy)benzoic acid(170 mg, 0.39 mmol) was refluxed with oxally chloride (1 mL) in dryCH₂Cl₂ (10 mL) and one drop of DMF for 3 h. The excess oxally chlorideand solvent were removed by distillation and the residue was dried undervacuum for 3 h and dissolved in CH₂Cl₂ (2 mL). This solution was addeddropwise to a solution of (+)-#i (150 mg, 0.20 mmol) and DMAP (75 mg,0.62 mmol) in CH₂Cl₂ (15 mL) at 0° C. The mixture was stirred at rtovernight, then saturated NaHCO₃ aqueous solution was added. The organiclayer was separated, and the aqueous layer was extracted with CH₂Cl₂.The organic phases were combined, dried (MgSO₄) and evaporated. Theresidue was purified by flash chromatography on silica gel (5%EtOAc/benzene) to afford the desired compound (215 mg, 91%).Recrystallization in CHCl₃ and ether gave a white powder: mp 52-54° C.;[α]_(D)=+37.5 (c=1.0, CHCl₃); ¹H NMR (CDCl₃, 400 MHz): δ 7.39-7.10 (m,42 H), 6.99 (t, J=7.4 Hz, 1 H), 6.93 (AB, J=8.3 Hz, 2 H), 6.31 (s, 1H)<5.65 (bs, 1 H), 5.13 (bs, 1 H), 5.07 (s, 2 H), 5.02 (s, 4 H), 5.00(s, 2 H), 4.92 (s, 4 H), 4.83 (AB, J=11.8 Hz, 2 H), 4.11 (s, 1 H), 3.15(bs, 1 H); ¹³C NMR (CDCl₃, 400 MHz): δ 165.2, 156.0, 155.9, 153.2,152.3, 148.8, 148.6, 142.6, 142.2, 137.4, 137.2, 137.0, 136.5, 131.4,128.7, 128.6, 128.5, 128.4, 128.2, 128.0, 127.9, 127.7, 127.3, 127.2,125.4, 125.1, 119.4, 114.6, 113.2, 110.3, 109.2, 101.0, 91.2, 75.1,71.1, 70.5, 70.0, 68.6, 28.7, 26.2; HRMS (ESI) calcd for C₇₈H₆₆O₁₀Na(M+Na) 1185.4554, found 1185.4542.

(−)-(2S,3R)-5,7-Bis(benzyloxy)-8-benzyl-2-[3,4-bis-(benzyloxy)-phenyl]chroman-3-yl3,4,5-tris(benzyloxy)-benzoate ((−)-#k)

Following the procedure used for the preparation of (−)-#j but with(−)-#g as starting material, (−)-(2S,3R)-5,7-bis(benzyloxy)-8-benzyl-2-[3,4-bis(benzyl-oxy)phenyl]-chroman-3-yl3,4,5-tris(benzyloxy)benzoate was obtained (90% yield) as a white solid:mp 103-105° C., [α]_(D)=−9.8 (c=1.3, CHCl₃); ¹H NMR (CDCl₃, 400 MHz): δ7.37-7.11 (m, 42 H), 6.90 (s, 1 H), 6.77 (m, 2 H), 6.27 (s, 1 H), 5.42(m, 1 H), 5.18 (d, J=6.3 Hz, 1 H), 5.06 (s, 4 H), 5.05-4.99 (m, 4 H),4.97 (s, 4 H), 4.88 (s, 2 H), 4.12 (AB, J=14.1 Hz, 2 H), 3.01 (a of ABt,J=16.8, 5.0 Hz, 1 H), 2.89 (B of ABt, J=16.8, 6.4 Hz, 1 H); ¹³C NMR(CDCl₃, 400 MHz): δ 156.0, 155.9, 155.4, 152.5, 152.3, 148.8, 148.6,142.3, 142.1, 137.3, 137.1, 137.0, 136.9, 136.5, 131.3, 128.7, 128.5,128.4, 128.3, 128.1, 128.0, 127.9, 127.8, 127.7, 127.5, 127.3, 127.2,127.1, 125.3, 124.9, 114.6, 112.8, 110.2, 108.9, 101.5, 91.0, 78.0,75.0, 71.2, 71.1, 71.0, 70.4, 70.0, 69.9, 28.6, 24.0; HRMS (ESI) calcdfor C₇₈H₆₆O₁₀Na (M+Na) 1185.4554, found 1185.4573.

(+)-(2S, 3S)-5,7-dihydroxy-8-benzyl-2-[3,4-dihydroxy-phenyl]chroman-3-yl3,4,5-trihydroxybenzoate ((+)-#I)

Under an H₂ atmosphere, Pd(OH)₂/C (20%, 400 mg) was added to a solutionof (+)-#j (200 mg, 0.17 mmol) in a sovlent mixture of THF/MeOH (1:1 v/v,20 mL). The reaction mixture was stirred at rt under H₂ for 6 h when TLCshowed that the reaction was completed. The reaction mixture wasfiltered to remove the catalyst. The filtrate was evaporated, and theresidue was rapidly purified by flash chromatograph on silica gel (10%MeOH/CH₂Cl₂, then 20% MeOH/CH₂Cl₂) to afford (+)-8-benzylcatechingallate ((+)-#1) (82 mg, 90% yield): mp 243-245° C. (decomposed);[α]_(D)=+123 (c=1.8, acetone); ¹H NMR (acetone-d₆, 400 MHz): δ 7.53 (d,J=7.4 Hz, 2 H), 7.42 (t, J=7.6 Hz, 2 H), 7.26-7.21 (m, 4 H), 7.04 (AB,J=8.2 Hz, 2 H), 6.31 (s, 1 H), 5.75 (bs, 1 H), 5.32 (bs, 1 H), 4.22 (AB,J=14.3 Hz, 2 H), 3.29 (a of ABt, J=17.4, 4.4 Hz, 1 H), 3.17 (b of AB,J=17.4 Hz, 1 H); ¹³C NMR (acetone-d₆, 400 MHz): δ 165.2, 154.2, 154.1,153.6, 144.9, 144.6, 144.3, 142.6, 137.9, 130.6, 128.4, 127.8, 125.0,120.9, 118.1, 114.7, 113.7, 109.1, 106.7, 98.0, 95.3, 77.1, 68.3, 25.9;HRMS (ESI) calcd for C₂₉H₂₄O₁₀Na (M+Na) 555.1267, found 555.1279.

(−)-(2S, 3R)-5,7-dihydroxy-8-benzyl-2-[3,4-dihydroxy-phenyl]chroman-3-yl3,4,5-trihydroxybenzoate ((−)-16)

Following the procedure for the preparation (+)-#I, but with (−)-#k asstarting material, (−)-8-benzylcatechin gallate ((−)-16) was obtained(91% yield): mp 239-241° C. (decomposed); [α]_(D)=−35 (c=2.0, acetone);¹H NMR (acetone-d₆, 400 MHz): δ 7.44 (d, J=7.1 Hz, 2 H), 7.33 (t, J=7.5Hz, 2 H), 7.23 (s, 2 H), 7.21 (t, J=7.3 Hz, 1 H), 6.92-6.86 (m, 2 H),6.34 (s, 1 H), 5.56 (m, 1 H), 5.35 (d, J=6.2 Hz, 1 H), 4.17 (AB, J=14.3Hz, 2 H), 3.12 (A of ABt, J=16.5, 5.2 HZ, 1 H), 2.98 (b of ABt, J=16.5.6.2 HZ, 1 H); ¹³C NMR (acetone-d₆, 400 MHz): δ 165.0, 154.1, 153.7,152.8, 144.9, 144.7, 144.6, 142.2, 137.8, 130.2, 128.3, 127.5, 124.8,120.5, 117.9, 114.8, 113.2, 108.8, 106.2, 98.2, 95.1, 77.7, 69.4, 23.5;HRMS (ESI) calcd for C₂₉H₂₄O₁₀Na 555.1267, found 555.1285.

Stability Tests of (−)-EGCG and 1

(−)-EGCG or 1 (0.1 mM) was incubated with RPMI 1640 culture medium at37° C. At different time points, 15 μL of the medium was injected intoan HPLC equipped with a C-18 reverse phase column (CAPCELL PAK C18 UG120, Shiseido Co., Ltd., 4.6 mm i.d. ×250 mm); flow rate, 1 mL/min;detection, UV 280 nm; for (−)-EGCG, time points were 0, 10, 20, 40, 60,90, 120 minutes and the mobile phase, 20% aqueous acetonitrile and 0.01%TFA; for prodrug 1, time points were 0, 30, 60, 90, 120 minutes andmobile phase, 50% aqueous acetonitrile and 0.01% TFA.

Enzymatic Hydrolysis of 1

Lysis buffer (pH 5) (0.25 mL) was added to 2×106 Jurkat T cells. Thiscould break the cell membrane of the cells and release the cytoplasmicenzymes. PBS (0.75 mL) was added which neutralized the medium to theoptimum pH value (pH 7) for the enzymes. Prodrug 1 (0.25 mM) was addedinto the reaction mixture and incubated at 37° C. At different timepoints (0, 30, 60, 90, 120, 150, 180, 210, 240, 300 and 360 minutes), analiquot (0.06 mL) of the reaction mixture was taken out, filtered andinjected into the HPLC and analyzed as outlined above.

Hydrolysis of 1 in the Presence of Vitamin C in Culture Medium With orWithout Lysates

Compound 1 (35 μM) was incubated with dulbecco's modified eagle medium(DMEM) (1 mL containing 1.67 mg/mL vitamin C) at 37° C. At differenttime points, 10 μL of the solution was injected into an HPLC equippedwith a C-18 reverse phase column; flow rate, 1 mL/min; detection, UV 280nm; mobile phase, 0-8 minutes (20% aqueous acetonitrile and 0.016% TFA),8-13 minutes (varying from 20% aqueous acetonitrile with 0.016% TFA to60% aqueous acetonitrile with 0.008% TFA).

For the investigation of hydrolysis of 1 in the presence of lysates,same concentration of 1 was incubated with DMEM (2 mL containing 1.67mg/mL vitamin C) in the presence of the lysates (5×105 breast cancercells with 0.15 mL lysis buffer). At different time points, an aliquot(0.06 mL) of the reaction mixture was taken out, filtered, injected intothe HPLC and analyzed as outlined above.

Cell Cultures

Human Jurkat T and LNCaP cells were cultured in RPMI supplemented with10% (v/v) fetal bovine serum, 100 U/ml penicillin, and 100 μg/mlstreptomycin. The non-transformed natural killer cells (YT line) weregrown in RPMI medium containing with 10% (v1v) fetal bovine serum, 100U/ml penicillin, 100 μg/ml streptomycin, 1 mM MEM sodium pyruvate, and0.1 mM MEM nonessential amino acids solution. Human breast cancer MCF-7cells, normal (WI-38) and simian virus-transformed (VA-13) humanfibroblast cells were grown in Dulbecco's modified Eagle's mediumssupplemented with 10% (v1v) fetal bovine serum, 100 U/ml penicillin, 100pg/ml streptomycin. All cell cultures were maintained in a 5% CO₂atmosphere at 37° C.

Cell Extract Preparation and Western Blotting

Whole cells extracts were prepared as described previously (An B,Goldfarb RH, Siman R, Dou QP. Novel dipeptidyl proteasome inhibitorsovercome Bc1-2 protective function and selectively accumulate thecyclin-dependent kinase inhibitor p27 and induce apoptosis intransformed, but not normal, human fibroblasts. Cell Death Differ 1998;5: 1062-75.). Analysis of Bax, IKBa, p27, PAW, and ubiquitinated proteinexpression were performed using monoclonal or polyclonal antibodiesaccording to previously reported protocols (An B et. al.).

Inhibition of Purified 20s Proteasome Activity by (−)-EGCG or SyntheticTea Polyphenols

Measurement of the chyrnotrypsin-like activity of the 20s proteasome wasperformed by incubating 0.5 μg of purified rabbit 20s proteasome with 40μM fluorogenic peptide substrate, Suc-Leu-Leu-Val-Tyr-AMC, with orwithout various concentrations of natural and synthetic tea polyphenolsas described previously (Nam S et. al.).

Inhibition of Proteasome Activity in Intact Cells by Natural/SyntheticTea Polyphenols

VA-13 or WI-38 cells were grown in 24 well plates (2 ml/well) to 70-80%confluency, followed by 24 h treatment with 25 pM (−)-EGCG, 2, or 2a. 40pM Suc-Leu-Leu-Val-Tyr-AMC substrate was then added for 2.5 h at 37° C.and the chyrnotrypsin-like activity was measured as described above.Alternatively, cells were treated with each compound at 25 μM for 4 or24 h, and harvested and lysed. Suc-Leu-Leu-Val-Tyr-AMC (40 μM) was thenincubated with the prepared cell lysates for 2.5 h and thechymotrypsin-like activity was measured as described above.

Immunostaining of Apoptotic Cells with Anti-Cleaved PARP Conjugated toFITC

Immunostaining of apoptotic cells was performed by addition of aFITC-conjugated polyclonal antibody that recognizes cleavedpoly(ADP-ribose) polymerase (PARP) and visualized on an Axiovert 2 5(Zeiss; Thornwood, NY) microscope. Cells were grown to about 80%confluency in 60 mm dishes. VA-13 cells were then treated with VP-16, 2,or 2a (25 μM) for 24 h. Following treatment, both suspension andadhering cells were collected and washed twice in PBS pH 7.4. The cellswere washed between all steps listed below and all washes are 1 minduration with PBS. Cells were then fixed in ice-cold 70% ethanol,permeabilized in 0.1% Triton-X-100 and blocked for 30 min in 1% bovineserum albumin (BSA) at room temperature. Incubation with the primaryFITC-conjugated-p85/PARP antibody was for 30 min at 4° C. in the darkwith mild shaking. Cell suspension was then transferred to glass slidesin the presence of Vector Shield mounting medium with DAPI. Images werecaptured using AxioVision 4.1 and adjusted using Adobe Photoshop 6.0software. Cell death was quantified by counting the number of apoptoticcells over the total number of cells in the same field. Data are mean ofduplicate experiments ±SD.

Trypan Blue Assay

Trypan blue assay was used to ascertain cell death in Jurkat T cellstreated with natural and synthetic polyphenols as indicated. Apoptoticmorphology was assessed using phase-contrast microscopy as describedpreviously (Kazi A, Hill R, Long T E, Kuhn D J, Twos E, Dou Q P. NovelN-thiolated beta-lactam antibiotics selectively induce apoptosis inhuman tumor and transformed, but not normal or nontransformed, cells.Biochem Pharmacol 2004;67:365-74; Kuhn D J, Smith D M, Pross S,Whiteside T L, Dou Q P. Overexpression of interleukin-2 receptor alphain a human squamous cell carcinoma of the head and neck cell line isassociated with increased proliferation, drug resistance, andtransforming ability. J Cell Biochem 2003;89:824-36).

MTT Assay

MTT was used to determine effects of polyphenols on overallproliferation of tumor cells. Human breast MCF-7 cells were plated in a96 well plate and grown to 70-80% confluency, followed by addition ofanalogs for 24 h. MTT (1 mg/ml) in PBS was then added to wells andincubated at 37° C. for 4 hours to allow for complete cleavage of thetetrazolium salt by metabolically active cells. Next, the MTT wasremoved and 100 μl of DMSO was added and colorimetric analysis performedusing a multilabel plate reader at 560 nm (Victor³; Perkin Elmer).Absorbance values plotted are the mean from triplicate experiments.

Soft Agar Assay

LNCaP cells (2×10⁴) were plated in soft agar on 6 well plates in thepresence of (−)-EGCG or protected tea analogs (25 pM) or in DMSO(control) to determine cellular transformation activity as describedpreviously (Kazi A et al).

Nuclear Staining

After each drug treatment, both detached and attached VA-13 and WI-38cells were stained Hoechst 33342 to assess for apoptosis. Briefly, cellswere washed 2× in PBS, fixed for 1 h with 70% ethanol at 4° C., washed3× in PBS, and stained with 50 pM Hoechst for 30 min in the dark at roomtemperature. Detached cells were plated on a slide and attached cellswere visualized on the plate with a fluorescent microscope at 10× or 40×resolution (Zeiss, Germany). Digital Scientific obtained images withAxioVision 4.1 and adjusted using Adobe Photoshop 6.0.

Induction of Caspase-3 Activity by Synthetic Tea Polyphenols

Cells were treated with each compound at 25 μM for 4 or 24 h, and thenharvested and lysed. Ac-DEVD-AMC (40 μM) was added to the cell lysatesfor 2.5 h and the caspase-3 activity.

Results Chemical Stability and Enzymatic Hydrolysis of Peracetate(−)-EGCG, 1

The stability of 1 with (−)-EGCG in a culture medium (RMPI) is compared,which mimics the body fluid with a pH value around 8. (−)-EGCG or 1 at0.1 mM was incubated in 1 mL RMPI at 37° C. at indicated times. Atdifferent time points, the medium was analyzed by HPLC for the amount oftested compound remaining. Degradation curves are shown in FIG. 2.

When (−)-EGCG was dissolved in the culture medium, it was found todegrade rapidly within 20 minutes, demonstrating the low stability of(−)-EGCG in the medium. Although 1 also degraded in the medium, as seenin FIG. 2, the rate of its degradation was much slower when comparedwith (−)-EGCG. 1 disappeared completely after 2 hours, indicating thatit is 6 times more stable than (−)-EGCG in this medium. Therefore,peracetate protection of the phenol groups of (−)-EGCG assists instabilizing 1 in culture (presumably physiological) conditions.

In order to determine if 1 was hydrolysed to EGCG under the culturemedium conditions, the experiment was repeated but now with addedvitamin C (at 1.67 mg/mL) to prevent the rapid degradation of thegenerated EGCG. As 1 disappeared, a new peak A was observed by HPLC toincrease and then decline in intensity with time. This was followed bythe appearance of another peak B in the HPLC which also eventuallydeclined. Finally, a peak in the HPLC identical in retention time toEGCG was observed to be formed. The time course results of thesecomponents were shown in FIG. 3. The identity of EGCG was confirmed byUV spectroscopy as well as mass spectrometry. Furthermore, massspectrometric analyses of peaks A and B showed that they were thedi-acetate and mono-acetate of EGCG respectively. These resultssuggested that compound 1 was first hydrolysed to the diacetate, thenmono-acetate and eventually EGCG under the culture medium conditions.

The generation of (−)-EGCG from compound 1 under cellular conditionscould be more clearly demonstrated by the addition of vitamin C toprevent the rapid disappearance of (−)-EGCG. In this case, we performedthe experiment in medium with breast cancer cell lysate. HPLC analysesshowed the disappearance of 1, together with the transient formation ofA (the di-acetate), B (the mono-acetate) and then (−)-EGCG in atime-course results (FIG. 4) similar to FIG. 3. Therefore, it isbelieved that that in medium with the addition of lysate, compound 1underwent hydrolysis forming the di-acetate of EGCG, then themono-acetate, then EGCG, and eventually gallic acid. (Scheme 1)

Inhibition of the Proteasomal Activity In Vitro and In Vivo by 1 and(−)-EGCG

If 1 is to function as the prodrug of (−)-EGCG, it should remainbiologically inactive until de-acetylation inside the cell where it isconverted into its parent compound. In order to test this hypothesis,proteasome activity was tested both in vitro and in intact Jurkat Tcells with either 1 or (−)-EGCG (as a positive control). First, 1 andcommercial (−)-EGCG were dissolved in DMSO and their effects on thechymotrypsin-like activity of purified 20S proteasomes were measured. At10 μM, 1 was completely inactive in inhibiting the chymotrypsin-likeactivity of the purified 20S proteasome (FIG. 5). In contrast, (−)-EGCGat 10 μM inhibited 80-90% of the proteasomal chymotrypsin-like activity.Therefore, as predicted, 1 outside of a cellular system is not aproteasome inhibitor.

If 1 converts to (−)-EGCG inside the cells, proteasome inhibition invivo should be detected. To examine this possibility, human Jurkat cellswas treated with 25 μM of 1 or (−)-EGCG for 12 or 24 h, followed bymeasurement of proteasome activity by using a chymotrypsin-like specificfluorogenic substrate in intact cells (FIG. 6 a) or Western blot forubiquinated proteins (FIG. 6 b). Treatment of Jurkat T cells with(−)-EGCG for 24 h inhibited proteasome activity by 31% versus 42%inhibition with 1 (FIG. 6 a). To analyze the intracellular level ofpolyubiquitinated proteins, cells were lysed after 12 hours incubationand subjected to Western blotting. 1 showed comparable levels ofubiquitinated proteins to that of natural (−)-EGCG (FIG. 6b). Therefore,1 is equally potent to, if not more potent than, (−)-EGCG in inhibitingthe proteasomal activity in intact cells. On the other hand, even though1 is six times more stable compared with EGCG, the potency of itsbiological activities in cells did not increase to a similar extent. Itis possible that the amount of EGCG generated from 1, and thus itsbiological activity inside the cells depends on a combination offactors: the relative permeability of 1 into the cells, the amount ofesterase enzymes and the amount of anti-oxidants that may be present inthe cells at any time.

Dephosphorylation of Akt in Jurkat Cells by 1 and (−)-EGCG

(−)-EGCG and 1 were incubated with Jurkat T cells for 24 h at 5, 10 and25 μM, followed by Western blot analysis using a specific antibody tophosphorylated Akt (FIG. 7). (−)-EGCG at 25 μM was found to reduce thelevel of p-Akt by 32% compared to treatment with 1, which lead to a 73%decrease in activated Akt at 25 μM as indicated by densitometricanalysis (FIG. 7). Actin was used as a loading control.

Cell Death Induced by 1 and (−)-EGCG

The abilities of (−)-EGCG and 1 to induce cell death in Jurkat T cellstreated with 10 μM for 24 h are also accessed. While (−)-EGCG had aminimal effect on cell death (5%), 1 was capable of inducing up to 15%cell death at that concentration (FIG. 8). Therefore, the greaterabilities of 1 to inhibit cellular proteasome activity (FIG. 6) and toinactivate Akt (FIG. 7) are associated with its increased celldeath-inducing activity (FIG. 8).

Acetylated Synthetic Tea Polyphenols Do Not Inhibit the Purified 20sProteasome Activity

Up to 25 μM of all protected and unprotected compounds were incubatedwith a purified 20s proteasome and a fluorogenic substrate forchyrnotrypsin activity for 30 min. The half-maximal inhibitoryconcentration or IC₅₀ was then determined. (−)-EGCG showed to be themost potent with an IC₅₀ of 0.2 μM, followed by 2 (IC₅₀ about 9.9 μM).IC₅₀ values of compounds 3 and 4 were found to be 14-15 μM. In contrast,the protected analogs were much less active: <35% inhibition at 25 μM.This is consistent with the results above.

Protected Tea Analogs Exhibit Greater Proteasome-Inhibitory Potency inIntact Tumor Cells

To determine what effects the synthetic tea analogs had on theproteasome in vivo, Jurkat cells were treated with 25 μM of eachsynthetic compound for either 4 or 24 h, with (−)-EGCG as a control(FIGS. 9A and 9B). After 4 h of treatnent, Western blot analysis showsthat the acetate-protected analogs induced a greater amount ofubiquitinated proteins (FIG. 9A, Lanes 5, 7, and 9), indicating thatproteasome activity is abrogated. 1 was used as a control based on theabove previous data showing that peracetate-protected (−)-EGCG is morepotent than natural (−)-EGCG (FIG. 9A, Lanes 3 vs. 2). Additionally,Western blots for Bax and IκBα, known as two proteasome targets,revealed the disappearance of these proteins and the appearance of ahigher molecular weight band, which is speculated to bemulti-ubiquinated forms of the proteins (FIG. 9A, lanes 3, 5, 7, and 9).However, after 24 h treatment, (−)-EGCG and its unprotected analogsexhibited a greater amount of ubiquitinated proteins (FIG. 9B. Lanes 2,4, 6, and 8). This is consistent with the idea that protected analogsare potent inhibitors of the proteasome at an earlier time point andthat after 24 h of treatment the ubiquitinated proteins are beingdepleted by deubiquitinating enzymes. Accumulation of p27, anotherproteasome target, is also found in Jurkat cells treated with theprotected analogs 2a, 3a and 4a (FIG. 9B, Lanes 9, 5, 7). The putativeubiquitinated IκBα band is still found in cells treated with 2a, 3a, and4a (FIG. 9B, Lanes 9, 5, 7). The presumed ubiquinated IκBα band is nowabsent in the 24 h treatment, possibly due to deubiquitination (FIG.9B). Actin was used as a loading control in this experiment.

Protected Analogs Induce Greater Cell Death in Leukemic Cells

In a kinetics experiment using a pair of analogs, 4 and 4a, it was foundthat the unprotected analog 4 induced accumulation of ubiquitinatedproteins with highest expression after 8 hours of polyphenol treatment(FIG. 9C). Conversely, the protected 4a showed increased ubiquitinatedprotein accumulation as early as 2 h and lasting up to 8 h (FIG. 9C). Todetermine if acetate-protected analogs are potent proteasome inhibitorsin other cancer cell systems, prostate cancer LNCaP cells were treatedfor 24 h with 25 μM of (−)-EGCG, 1, 2a, or 3a, with DMSO as a control.Indeed, ubiquitin-conjugated proteins were observed, with the greatestincrease found in cells treated with 2a and 3a (FIG. 9D).

Protected Analogs are More Potent Apoptosis Inducers

It has been shown that proteasome inhibition can induce apoptosis in awide variety of cancer cells, but not in normal, non-transformed cells(An B et al). Jurkat T cells are treated with 25 μM of each of theselected polyphenols and their protected analogs for 24 h to investigatetheir abilities to induce apoptotic cell death. Trypan blueincorporation assay revealed that 2a, 3a and 4a, but not others, induceddeath in 99, 57, and 83% of Jurkat cells, respectively (FIG. 10A).Similarly, Western blot analysis showed that only 2a, 3a, and 4a inducedapoptosis-specific PARP cleavage after 24 h (FIG. 10B). Animmunofluorescent stain that detects only the cleavage PARP fiagment(p85; green) showed that SV40-transformed VA-13 cells are highlysensitive to apoptosis induced by 2a with 73% apoptotic cells after 24 htreatment (FIGS. 10C and 10D). The unprotected 2 induced much lessapoptosis (21%), while 25 μM VP-16, used as a positive control, induced92% apoptosis (FIGS. 10C and 10D). Counterstain with DAPI, which bindsto the minor groove in A-T rich regions of DNA, was decreaseddrastically in apoptotic cells (FIG. 10C), consistent with DNAfragmentation in late stage apoptosis.

Inhibition of Tumor Cell Proliferation by Protected Polyphenols

Treated breast cancer (MCF-7) cells were then treated with 5 or 25 μM ofperacetate-protected analogs for 24 h, followed by MTT analysis todetermine their effects on cell proliferation. Compound 1 at 25 μMinhibited cellular proliferation by 40% (FIG. 11A). The protectedcompounds 2a, 3a, and 4a caused 50% inhibition at 5 μM and 70% at 25 μM,respectively (FIG. 11A).

Human prostate cancer LNCaP cells were then treated for 24 h with eachselected tea polyphenol protected analogs 1, 2a, 3a, 4a at 25 μM,followed by determining the apoptotic morphological changes. Again, theprotected analogs 2a, 3a, and 4a caused dramatic round-up, detachment,and cellular fragmentation (FIG. 11B). 1 induced mild morphologicalchanges, while (−)-EGCG treatment led to enlarged, flattened cells,indicating growth arrest.

Soft agar assay is used to determine the transforming activity of tumorcells. Abrogation of colony formation is linked to GI arrest and/orapoptosis. LNCaP cells were added to soft agar in 6-well plates, andwere then treated one time at initial plating with 25 μM of (−)EGCG or aprotected analog (FIG. 11C). After 21 days, colony formation wasevaluated. Cells treated with (−)-EGCG showed a significant decrease incolony formation compared to the control cells treated with DMSO (FIGS.11C and 11D). Protected polyphenols also inhibited tumor celltransforming activity, with 2a and 4a being the most potent inhibitorsof colony formation (FIG. 11D).

Preferential Induction of Apoptosis in Tumor Cells by Protected Analogs

The ability to induce apoptosis in tumor cells, but not normal cells isan important measure for novel anti-cancer drugs. To determine whetherthe protected compounds also affect normal cells, both VA-13 and WI-38cells were treated with 25 μM of (−)-EGCG, 2, or 2a for 24 h andexamined proteasome activity, nuclear morphological changes, anddetachment. A differential decrease was found in the chyrnotrypsin-likeactivity of the proteasome in VA-13 cells over normal WI-38 cells (FIG.12A). A 42-48% decrease in proteasome activity was observed in VA-13cells treated with (−)-EGCG and 2, while 2a inhibited 92% of theproteasomal activity. Conversely, the proteasome activity in WI-38 cellswas decreased by only −5% with all three polyphenol treatments.

Next, apoptotic nuclear morphology is examined after treatment withnatural (−)-EGCG, 2, and 2a (FIG. 12B). While (−)-EGCG and 2 exhibitedlittle or no apoptosis, 2a markedly induced apoptosis inSV-40-transformed VA-13 cells. In contrast, normal WI-38 fibroblaststreated with all the compounds did not undergo apoptosis and very littledetachment was visible. A comparison among all the protected analogs wasthen performed using 25 μM for 24 h. After 36 h, (−)-EGCG did initiateapoptosis in VA-13 cells (FIG. 12C). All of the protected analogsinduced apoptosis in transformed (VA-13), but not normal (WI-38) cells(FIGS. 12B and 12C). Similarly, when leukemic (Jurkat T) and normal,non-transformed natural killer (YT) cells were treated with (−)-EGCG and2a for 24 h, only Jurkat cells underwent apoptosis as evidenced by PAWcleavage (FIG. 12D).

Inhibition of Purified 20S Proteasome Activity by New Synthetic TeaPolyphenol Analogs.

The green tea polyphenol (−)-EGCG contains a B-ring with three —OHgroups (FIG. 1) and has an IC₅₀ value of 0.3 μM to a purified 20Sproteasome (Table 1).

TABLE 1 Inhibition of proteasome activity to synthetic teapolyphenols^(1,2) Unprotected IC₅₀ (μM) Protected IC₅₀ (μM) (−)-EGCG0.30 ? 0.02 No 16 0.59 ? 0.17 No 24* n.a.³ No 6 2.69 ? 1.07 No 19* n.a.³No 15 4.56 ? 1.20 No 12 5.99 ? 0.13 No 23* n.a.³ No 11 7.70 ? 0.14 No22* n.a.³ No 10 8.51 ? 0.47 No 21* n.a.³ ¹Inhibition of purified 20Sproteasome was assessed using a chymotrypsin-like specific fluorogenicsubstrate (Suc-Leu-Leu-Val-Tyr-AMC). 20S proteasomes were incubated withpolyphenols for 2 h. ²Results obtained from 3 independent experimentsperformed in triplicate. ³n.a. indicates that the inhibitory activity ofthe purified 20S proteasome at 25 μM was <20%.

Removal of an —OH from B-ring of (−)-EGCG generates (−)-ECG that hasdecreased proteasome-inhibitory potency in vitro (0.58 μM). To furtherexamine the effects of B-ring —OH group deletion onproteasome-inhibitory and cell death-inducing abilities, we synthesizedseveral novel EGCG analogs with eliminated —OH groups on the B-ring aswell as their putative prodrugs, the peracetate-protected counterparts.By using a purified 20S proteasome and a chymotrypsin-like fluorogenicsubstrate, the proteasome-inhibitory effects of the followingunprotected polyphenol analogs are determined:

TABLE 2 Effects of natural and synthetic tea polyphenols on 20Sprokaryotic proteasome activities. Reference a is Smith, D. M.; Daniel,K. G.; Wang, Z.; Guida, W. C.; Chan, T. H.; Dou, Q. P. Proteins:Structure, Function, and Bioinformatics, 2004, 54, 58 Compound IC50 (μM)Reference (−)-EGCG 0.30 This work (−)-ECG, 8 0.58 Reference a (+)-CG, 70.73 Reference a (−)-EZG, 6 2.69 This work (+)-ZG, #m 4.56 This work #n0.84 This work #o 1.22 This work 15 4.56 This work (+)-ECG 0.73Reference a #1 0.39 This work (−)-CG 0.75 Reference a 16 0.59 This work

A good correlation between proteasome-inhibitory activity and the numberof —OH groups on the B-ring and on the D-ring is observed. As individual—OH groups were eliminated from the B-ring, the IC₅₀ values increasedstepwise and the order of potency observed was: (−)-EGCG (IC₅₀ 0.30μM)>(−)-ECG (0.58 μM)>6 (2.69 μM)>#m (4.56 μM) (FIG. 13 and Table 1).Consistently, in the series of compounds 12, 11 (an epimer of 12) and 10which contain one —OH on the D-ring (FIG. 13), 12 and 11 with two B-ring—OH groups are more potent than 10 with only one B-ring —OH group (5.99,7.70, and 8.51 μM, respectively; Table 1).

Additional SAR analysis also revealed that when more —OH groups wereremoved from the D-ring, the inhibitory potency was further decreased ascompared to B-ring —OH eliminations alone. For example, 6 is more potentthan 10 (2.69 vs. 8.51 μM), and (−)-ECG as well as (−)-CG are morepotent than No 12 [0.58 and 0.75 vs. 5.99 μM;]. Finally, we haveincluded compound 16 to probe the hydrophobic effect around the A ring.Indeed, compound 16 was found to be more active than the counterpart(−)-CG [0.59 vs. 0.75 μM; FIG. 13; Table 1 and 2].

As a comparison, the inhibitory activities of the protected polyphenolanalogs to purified proteasome are also determined. As expected, theperacetate-protected analogs (indicated by *) were not potent inhibitorsof the chymotrypsin-like activity of the purified 20S proteasome,compared to their unprotected analogs, perhaps due to the lack ofcellular esterases required for conversion. All the protected analogs at25 μM inhibited <20% of the purified 20S proteasome activity (Table 1).

Inhibition of Cellular Proteasome Activity by Synthetic Tea Polyphenols

To determine whether the unprotected and protected polyphenol analogscould inhibit the proteasomal activity in intact cells, leukemia JurkatT cells were treated with each compound at 25 μM for 4 and 24 h,followed by collection of cell pellets and measurement of theproteasomal chymotrypsin-like activity in the prepared lysates. Theunprotected compounds show limited proteasome inhibition in intactJurkat T cells although 10 and 15 showed moderate potency (FIG. 14).These data suggest that most, if not all, of the unprotected compoundsare only moderately active in the cellular environment, similar to(−)-EGCG, presumably because of their cellular instability.

Importantly, all of the peracyloxyl-protected, particularly,peracetate-protected polyphenols tested were much more potent inhibitorsof the proteasomal chymotrypsin-like activity than their unprotectedcounterparts after 24 h treatment (FIG. 14). Especially, 19* inhibited97% of the chymotrypsin-like activity while its unprotected polyphenolcounterpart, 6, inhibited only 30% of the chymotrypsin-like activityafter a 24 h treatment.

The pro-apoptotic protein Bax and the cyclin-dependent kinase inhibitorp27 are natural proteasome targets. If the peracetate-protectedpolyphenols inhibit the intact proteasome activity, we would expectaccumulation of Bax and p27 in these cells. To test this idea, Jurkat Tcells were treated with both protected and unprotected compounds andlysates were analyzed by Western blot using specific antibodies to Baxand p27 (FIG. 15). After 4 h of treatment, the peracetate-protectedanalogs 24* and 19*, but not their unprotected analogs, were able toincrease accumulation of both Bax (by 3- and 2.5-fold, respectively) andp27 (6- and 11-fold, respectively) (FIG. 15A). After 24 h treatment, Baxlevels were increased in cells treated with all of theperacetate-protected compounds (13-, 12-, 40- and 36-fold, respectively,by 24*, 19*, 21* and 23*). However, p27 increase was observed withtreatment of mainly 21* (2-fold) and 23* (2-fold) after 24 h (FIG. 15B).In addition, levels of ubiquitinated proteins, an indicator ofproteasome inhibition, were significantly increased by the protected22*, but not its corresponding, unprotected 11, after 4 h treatment(FIG. 15C). These data are consistent with the profile of theproteasome-inhibitory activities of these compounds (FIG. 14). It issuggested the protected analogs can be converted into active proteasomeinhibitors in cultured tumor cells.

Induction of Apoptotic Cell Death by Synthetic Tea Polyphenol Analogs.

It has previously been shown that proteasome inhibition is associatedwith apoptosis induction. To determine if the B-ring/D-ring protectedanalogs were capable of inducing cell death, Jurkat T cells were firsttreated with all the protected and unprotected analogs at 25 μM for 24h, followed by trypan blue exclusion assay (FIG. 16). Changes in cellmorphology (shrunken cells and characteristic apoptotic blebbing) wereobserved after 4 h treatment with both protected and unprotected analogsand these changes were greatly increased after 24 h treatment withprotected analogs (data not shown). Furthermore, theperacetate-protected analogs induced a 5- to 10-fold increase in celldeath after 4 h incubation and up to 20-fold increase at 24 h (FIG. 16).In contrast, the unprotected polyphenols caused much less cell death,only 3- to 5-fold increase after even 24 h treatment (FIG. 16).

To determine if the cell death observed (FIG. 16) was due to theinduction of apoptosis, a similar experiment was performed in Jurkat Tcells, followed by measurement of caspase-3 activation and PARPcleavage. The peracetate-protected polyphenols induced the highestlevels of caspase-3 activity (FIG. 17A): 24* treatment for 4 h resultedin an 16-fold increase while the unprotected 16 resulted in only a4-fold increase. Furthermore, the protected 19* treatment increased thecaspase-3 activity by 16-fold, while the unprotected counterpart, 6, hadmuch less effect (4-fold). Consistently, PARP cleavage was detected incells treated with 24* and 19*, but not with the unprotectedcounterparts 16 and 6 (FIG. 17B). Furthermore, the protected compound22*, but not its unprotected counterpart 11, was able to induce PARPcleavage in cells after 4 h treatment (FIG. 17C).

Preferential Induction of Apoptosis in Tumor Cells byPeracetate-Protected (−)-EGCG Analogs.

Apoptosis induction in tumor cells but not in normal cells is animportant aspect of anti-cancer drugs. To determine whether theperacetate-protected analogs affect normal cells, we treated bothleukemic Jurkat T cells and non-transformed immortalized natural killer(YT) cells with the seemingly most potent peracetate-protected analog,19*. Again, levels of proteasome target proteins Bax, p27 (data notshown), and IκBα (FIG. 18) were increased in leukemia Jurkat T cells.However, levels of these proteins were not increased in thenon-transformed immortalized YT cells. In addition, levels ofubiquitinated proteins were accumulated in a dose-dependent manner inJurkat T cells. In contrast, only a slight, transient increase in levelsof ubiqitinated proteins was detected in the YT cells. Consistent withthe selective inhibition of the proteasome activity, theapoptosis-specific PARP cleavage was found only in the Jurkat T, but notYT cells. Thus, potent peracetate-protected polyphenols apparently donot induce proteasome inhibition and subsequent apoptosis innon-transformed YT cells.

Discussion

Natural (−)-EGCG from green tea has been converted to its peracetatecompound 1. In addition, several synthetic analogs to (−)-EGCG thatpossess deletions of the hydroxyl groups on the gallate ring aresynthesized. Additionally, the hydroxyl groups were converted to acetategroups to create a prodrug, which could be cleaved via esterases insidethe cell and converted to the parent drug. Surprisingly, the protectedanalogs were much more potent proteasome inhibitors in intact tumorcells than their unprotected counterparts. Consistently, the protectedanalogs were also more potent apoptosis inducers than unprotectedpartners, when tested in leukemic (Jurkat), solid tumor and transformedcell lines. SAR analysis of the protected analogs revealed that theorder of their potency is as follows: 2a=4a>3a>1>(−)-EGCG.

Protected analogs of (−)-EGCG were designed and synthesized.Unexpectedly, these compounds appear to have proteasome inhibitoryactivity in vivo.

The peracyloxyl, particularly peracetate-protected analogs are alsofound to be more potent proteasome inhibitors than their unprotected,hydroxylated counterparts. It was suggested that the N-terminal Thr ofthe chymotrypsin-like subunit (β5) of the proteasome executes anucleophilic attack on the ester-bonded carbon of (−)-EGCG, whichinitiates irreversible acylation to the β5-subunit and inhibits itsprotease activity (Nam S, Smith D M and Dou Q P: J Biol Chem 276:13322-13330, 2001.). However, addition of the peracetate moietiesreduces the electrophilic character of the ester-bond carbon, leading tomuch reduced inhibition to the purified 20S proteasome. Unsurprisingly,the protected polyphenols examined here also exhibited limitedinhibition to the chymotrypsin-like activity of purified 20S proteasome(Table 1).

A correlation is shown between the activity of proteasome inhibition andthe number of —OH groups on the B-ring (Table 2). The IC₅₀ values ofunprotected analogs increased stepwise as individual —OH groups wereeliminated (Table 1). Although, 16 contains an additional benzene ringoff the C₈ carbon on the A ring of (−)-CG, it has slightly increasedproteasome-inhibitory potency to (−)-CG (0.59 vs. 0.75 μM).

Another important SAR found is that further elimination of —OH groupsfrom the D-ring leads to further decreased proteasome-inhibitory potencyin vitro (Table 1).

As shown in FIG. 14, the peracetate-protected polyphenols were much morepotent inhibitors of the proteasomal chymotrypsin-like activity than theunprotected polyphenols. Peracetate-protected analogs appear to bestable for extended time points evidenced by the decrease in cellularproteasomal chymotrypsin-like activity at 24 h. In contrast, theunprotected analogs appear to lose stability after 24 h treatment.Cellular proteasomal chymotrypsin-like activity was inhibited 97% byanalog 19* after 24 h while its unprotected counterpart, 6, inhibitedonly 30% of the activity. Compared to other pairs of protected vs.unprotected compounds, 22* displayed relatively small increase inchymotrypsin-inhibitory potency from its counterpart 11, indicatingpossible conversion of 22* to the parent drug in cells. Although themechanism is not fully understood, other peracetate-protected green teaanalogs seem to be converted to distinct potent compounds in a cellularenvironment.

Several proteasome target proteins were also evaluated after proteasomeinhibition with synthetic tea polyphenols (FIG. 15). The data providedanother piece of evidence that peracetate-protected analogs require themilieu of the cell and/or cell extract to be converted into effectiveproteasome inhibitors, represented by the accumulation of Bax, p27, andubiquitinatied proteins. The protected analogs were able to accumulatetarget proteins with greater efficacy than their unprotectedcounterparts in a cellular model compared to the in vitro model. Incontrast, the unprotected analogs did not appear to accumulate targetproteins to the extent of the protected analogs, which may be a resultof proton donation from the —OH groups and subsequent degradation. Thedata further indicates that the peracetate-protected analogs undergoconversion to a new compound and act as prodrugs in vivo.

Trypan blue analysis confirmed the occurrence of cell death and thecharacteristic apoptotic morphological changes induced after treatmentwith mainly protected analogs (FIG. 16). The protected analogs againappeared more stable at 24 h (cell death greater than 50% in all) thanthe unprotected analogs. Caspase-3 activity confirms that apoptosisoccurred after 4 h incubation with the protected compounds; caspase-3activity was increased up to 16-fold compared to the control (FIG. 17).Western blot analysis for PARP cleavage further showed appearance after4 h treatment with the protected analogs such as 19*, 24*, and 22*. Mostimportantly, the peracetate-protected (−)-EGCG analog, 19*,preferentially induced apoptosis in Jurkat T cells while thenon-transformed YT cells remained less unaffected (FIG. 18), suggestinga potential for the protected analogs to be developed into novelanti-cancer agents.

In summary, epidemiological studies have suggested the anti-cancerbenefits of green tea consumption. The proteasome has been indicated inthe pathological state of cancer (Ciechanover A: Embo J 17: 7151-7160,1998.), and green tea polyphenols as inhibitors of the proteasome may bea viable treatment for some cancers. Our current study further suggestthat synthetic green tea polyphenols containing peracetate-protected —OHgroups are potent proteasome inhibitors and that further examination ofthese compounds may elucidate additional benefits from this form oftherapeutics.

This invention provides a variety of derivatives of (−)-EGCG that is atleast as potent as (−)-EGCG. The carboxylate protected forms of (−)-EGCGand its derivatives are found to be more stable than the unprotectedforms, which can be used as proteasome inhibitors to reduce tumor cellgrowth. Further, from the structures of 1, 2, 3, 4, 19*, 21*, 22* and23* it can be seen that some of the hydroxyl groups of the gallate ringof (−)-EGCG may not be important to the potency.

While the preferred embodiment of the present invention has beendescribed in detail by the examples, it is apparent that modificationsand adaptations of the present invention will occur to those skilled inthe art. Furthermore, the embodiments of the present invention shall notbe interpreted to be restricted by the examples or figures only. It isto be expressly understood, however, that such modifications andadaptations are within the scope of the present invention, as set forthin the following claims. For instance, features illustrated or describedas part of one embodiment can be used on another embodiment to yield astill further embodiment. Thus, it is intended that the presentinvention cover such modifications and variations as come within thescope of the claims and their equivalents.

1. A compound for inhibiting proteasome having the formula:

wherein R₁₁, R₁₂, R₁₃, R₂₁, R₂₂, R₂, R₃, and R₄ are each independentlyselected from the group consisting of —H, and C₁ to C₁₀ acyloxyl group;and R₅ is selected from the group consisting of —H, C₁-C₁₀-alkyl,C₂-C₁₀-alkenyl, C₂-C₁₀-alkynyl, C₃-C₇-cycloalkyl, phenyl, benzyl andC₃-C₇-cycloalkenyl, whereas each of the last mentioned 7 groups can besubstituted with any combination of one to six halogen atoms; at leastone of R₁₁, R₁₂, R₁₃, R₂₁, R₂₂, R₂, R₃, and R₄ is C₁ to C₁₀ acyloxyl andat least one of R₁₁, R₁₂, R₁₃, R₂₁, R₂₂, R₂, R₃, and R₄ is —H.
 2. Thecompound of claim 1, wherein each of R₁₁ R₂, and R₄ is —H, and each ofR₂, R₁₃, R₂₁, R₂₂, and R₃ is an acetate group.
 3. The compound of claim1, wherein each of R₁₁ R₂, and R₄ is —H, and each of R₁₂, R₁₃, R₂₁, R₂₂,and R₃ is a benzoate group.
 4. The compound of claim 1, wherein R₁₁ is—H, and each of R₁₂, R₁₃, R₂₁, R₂₂, R₂, R₃, and R₄ is an acetate group.5. The compound of claim 1, wherein R₁₁ is —H, and each of R₁₂, R₁₃,R₂₁, R₂₂, R₂, R₃, and R₄ is a benzoate group.
 6. The compound of claim1, wherein each of R₁₁, R₁₃, R₂, and R₄ is —H, and each of R₁₂, R₂₁,R₂₂, and R₃ is an acetate group.
 7. The compound of claim 1, whereineach of R₁₁, R₁₃, R₂, and R₄ is —H, and each of R₁₂, R₂₁, R₂₂, and R₃ isa benzoate group.
 8. The compound of claim 1, wherein each of R₁₁, andR₁₃ is —H, and each of R₁₂, R₂₁, R₂₂, R₂, R₃, and R₄ is an acetategroup.
 9. The compound of claim 1, wherein each of R₁₁, and R₁₃ is —H,and each of R₁₂, R₂₁, R₂₂, R₂, R₃, and R₄ is a benzoate group.
 10. Thecompound of claim 1, wherein each of R₁₁, R₁₂, and R₁₃ is —H, and eachof R₂, R₂₂, R₂, R₃, and R₄ is an acetate group.
 11. The compound ofclaim 1, wherein each of R₁₁, R₁₂, and R₁₃ is —H, and each of R₂₁, R₂₂,R₂, R₃, and R₄ is a benzoate group.
 12. The compound of claim 1, whereinR₅ is —H, and each of R₁₁, R₁₂, R₁₃, R₂₁, and R₂₂ is an acetate group.13. The compound of claim 12, wherein R₂ is an acetate group, and eachof R₃ and R₄ is —H.
 14. The compound of claim 12, wherein R₃ is anacetate group, and each of R₂ and R₄ is —H.
 15. The compound of claim12, wherein each of R₂ and R₄ is an acetate group, and R₃ is —H.
 16. Amethod of reducing tumor cell growth including the step of administeringan effective amount of a compound having the formula:

wherein R₁₁, R₁₂, R₁₃, R₂₁, R₂₂, R₂, R₃, and R₄ are each independentlyselected from the group consisting of —H, and C₁ to C₁₀ acyloxyl group;and R₅ is selected from the group consisting of —H, C₁-C₁₀-alkyl,C₂-C₁₀-alkenyl, C₂-C₁₀-alkynyl, C₃-C₇-cycloalkyl, phenyl, benzyl andC₃-C₇-cycloalkenyl, whereas each of the last mentioned 7 groups can besubstituted with any combination of one to six halogen atoms; and atleast one of R₁₁, R₁₂, R₁₃, R₂₁, R₂₂, R₂, R₃, and R₄ is C₁ to C₁₀acyloxyl
 17. The use of a compound of having the formula:

wherein R₁₁, R₁₂, R₁₃, R₂₁, R₂₂, R₂, R₃, and R₄ are each independentlyselected from the group consisting of —H, and C₁ to C₁₀ acyloxyl group;and R⁵ is selected from the group consisting of —H, C₁-C₁₀-alkyl,C₂-C₁₀-alkenyl, C₂-C₁₀-alkynyl, C₃-C₇-cycloalkyl, phenyl, benzyl andC₃-C₇-cycloalkenyl, whereas each of the last mentioned 7 groups can besubstituted with any combination of one to six halogen atoms; and atleast one of R₁₁, R₁₂, R₁₃, R₂₁, R₂₂, R₂, R₃, and R₄ is C₁ to C₁₀acyloxyl group in the manufacturing of a medicament for reducing tumorcell growth.
 18. A compound for inhibiting proteasome having theformula:

wherein R₁ is —H; R₂, R₃, and R₄ are each independently selected fromthe group consisting of —H and —OH; and at least one of R₂, R₃, and R₄is —H.
 19. The compound of claim 18, wherein R₂ is —OH, and each of R₃and R₄ is —H.
 20. The compound of claim 18, wherein R₃ is —OH, and eachof R₂ and R₄ is —H.
 21. The compound of claim 18, wherein each of R₂ andR₄ is —OH, and R₃ is —H
 22. A method of reducing tumor cell growthincluding the step of administering an effective amount of a compoundhaving the formula:

wherein R₁ is —H; R₂, R₃, and R₄ are each independently selected fromthe group consisting of —H and —OH; and if R₂═R₃═R_(4,) then R₂ is not—OH.
 23. The use of a compound of having the formula:

wherein R₁ is —H; R₂, R₃, and R₄ are each independently selected fromthe group consisting of —H and —OH; and if R₂═R₃═R₄, then R₂ is not —OHin the manufacturing of a medicament for reducing tumor cell growth.